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LOCUS Exported 8596 bp ds-DNA linear SYN 18-MAY-2021
DEFINITION 3rd generation lentiviral transfer vector. pPRIME cloning plasmid
with a CMV promoter controlling expression of dsRed and miR30-based
shRNA targeting firefly luciferase.
ACCESSION .
VERSION .
KEYWORDS pPRIME-CMV-dsRed-FF3
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8596)
AUTHORS Stegmeier F, Hu G, Rickles RJ, Hannon GJ, Elledge SJ
TITLE A lentiviral microRNA-based system for single-copy polymerase
II-regulated RNA interference in mammalian cells.
JOURNAL Proc Natl Acad Sci U S A. 2005 Sep 13. 102(37):13212-7.
PUBMED 16141338
REFERENCE 2 (bases 1 to 8596)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 18, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..8596
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 277..480
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 526..1203
/codon_start=1
/product="rapidly maturing tetrameric variant of DsRed
fluorescent protein (Bevis and Glick, 2002)"
/label=DsRed-Express
/note="mammalian codon-optimized"
/translation="MDSSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
VTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHH
LFL"
primer_bind complement(656..674)
/label=mCherry-R
/note="mCherry, reverse primer"
primer_bind complement(706..726)
/label=DsRed1-N
/note="DsRed1, reverse primer"
primer_bind 924..943
/label=mCherry-F
/note="mCherry, forward primer"
primer_bind 1118..1141
/label=DsRed1-C
/note="DsRed1, forward primer"
ncRNA 1253..1374
/label=5' miR-30a
/note="sequence upstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)"
/ncRNA_class="miRNA"
ncRNA 1401..1415
/label=miR-30a loop
/note="loop from the the 71-nt precursor of the human
miR-30a microRNA (Zeng et al., 2002)"
/ncRNA_class="miRNA"
ncRNA 1443..1569
/label=3' miR-30a
/note="sequence downstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)"
/ncRNA_class="miRNA"
promoter 1723..1825
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
CDS 1826..2485
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
primer_bind complement(1892..1911)
/label=CAT-R
/note="Chloramphenicol resistance gene, reverse primer"
CDS 2615..2644
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
primer_bind 2669..2685
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
misc_feature 2760..3348
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(2813..2833)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(3231..3242)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
primer_bind complement(3351..3367)
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
LTR 3877..4057
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
rep_origin complement(4119..4707)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(4199..4218)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(4878..5738)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind 5501..5520
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(5739..5843)
/gene="bla"
/label=AmpR promoter
primer_bind complement(5918..5937)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 6109..6488
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 6490..6688
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 6706..6886
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 6933..7058
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 7555..7788
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 7973..8017
/codon_start=1
/product="antigenic peptide corresponding to amino acids
655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik
et al., 2013)"
/label=gp41 peptide
/note="recognized by the 2H10 single-chain llama nanobody"
/translation="KNEQELLELDKWASL"
misc_feature 8315..8432
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
enhancer 8569..276
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
ORIGIN
1 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
61 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactacc
121 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
181 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
241 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
301 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
361 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
421 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
481 ggtttagtga accgtcagat ccgctagcgc taccgggcct gcaggatgga ctcctccgag
541 gacgtcatca aggagttcat gcgcttcaag gtgcgcatgg agggctccgt gaacggccac
601 gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag
661 ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc cccccagttc
721 cagtacggct ccaaggtgta cgtgaagcac cccgccgaca tccccgacta caagaagctg
781 tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg
841 accgtgaccc aggactcctc cctgcaggac ggctccttca tctacaaggt gaagttcatc
901 ggcgtgaact tcccctccga cggccccgta atgcagaaga agactatggg ctgggaggcc
961 tccaccgagc gcctgtaccc ccgcgacggc gtgctgaagg gcgagatcca caaggccctg
1021 aagctgaagg acggcggcca ctacctggtg gagttcaagt ccatctacat ggccaagaag
1081 cccgtgcagc tgcccggcta ctactacgtg gactccaagc tggacatcac ctcccacaac
1141 gaggactaca ccatcgtgga gcagtacgag cgcgccgagg gccgccacca cctgttcctg
1201 taggcggccg caagccttgt taagtgctcg cttcggcagc acatatacta tgttgaatga
1261 ggcttcagta ctttacagaa tcgttgcctg cacatcttgg aaacacttgc tgggattact
1321 tcttcaggtt aacccaacag aaggctcgag aaggtatatt gctgttgaca gtgagcgagc
1381 tcccgtgaat tggaatccta gtgaagccac agatgtagga ttccaattca gcgggagcct
1441 gcctactgcc tcggaattca aggggctact ttaggagcaa ttatcttgtt tactaaaact
1501 gaataccttg ctatctcttt gatacatttt tacaaagctg aattaaaatg gtataaatta
1561 aatcactttt ttcaattgga agactaatgc gtttaaacac gcggcgacgc gttcgaccga
1621 ataaatacct gtgacggaag atcacttcgc agaataaata aatcctggtg tccctgttga
1681 taccgggaag ccctgggcca acttttggcg aaaatgagac gttgatcggc acgtaagagg
1741 ttccaacttt caccataatg aaataagatc actaccgggc gtattttttg agttgtcgag
1801 attttcagga gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga
1861 tatatcccaa tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac
1921 ctataaccag accgttcagc tggatattac ggccttttta aagaccgtaa agaaaaataa
1981 gcacaagttt tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgga
2041 attacgtatg gcaatgaaag acggtgagct ggtgatatgg gatagtgttc acccttgtta
2101 caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga
2161 tttccggcag tttctacaca tatattcgca agatgtggcg tgttacggtg aaaacctggc
2221 ctatttccct aaagggttta ttgagaatat gtttttcgtc tcagccaatc cctgggtgag
2281 tttcaccagt tttgatttaa acgtggccaa tatggacaac ttcttcgccc ccgttttcac
2341 catgggcaaa tattatacgc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca
2401 tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg
2461 cgatgagtgg cagggcgggg cgtaattttt ttaaggcagt tattggtgcc cttaaacgcc
2521 tggttgctac gcctgaataa gtgataataa gcggatgaat ggcagaaatt cggatctcgc
2581 gcgtttgggc ggtggctccc tgccacgcgg ctccgaacag aagctgatct ccgaagagga
2641 tctgacatgt gtttaaactt aattaagtcg aggtcgacgg tatcgataag ctcgcttcac
2701 gagatcatgt ttaagggttc cggttccact aggtacaatt cgatatcaag cttatcgata
2761 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
2821 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
2881 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt
2941 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
3001 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta
3061 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt
3121 tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg
3181 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
3241 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
3301 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc
3361 gacctcgatc gagacctaga aaaacatgga gcaatcacaa gtagcaatac agcagctacc
3421 aatgctgatt gtgcctggct agaagcacaa gaggaggagg aggtgggttt tccagtcaca
3481 cctcaggtac ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta
3541 aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga tatccttgat
3601 ctgtggatct accacacaca aggctacttc cctgattggc agaactacac accagggcca
3661 gggatcagat atccactgac ctttggatgg tgctacaagc tagtaccagt tgagcaagag
3721 aaggtagaag aagccaatga aggagagaac acccgcttgt tacaccctgt gagcctgcat
3781 gggatggatg acccggagag agaagtatta gagtggaggt ttgacagccg cctagcattt
3841 catcacatgg cccgagagct gcatccggac tgtactgggt ctctctggtt agaccagatc
3901 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
3961 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
4021 ctcagaccct tttagtcagt gtggaaaatc tctagcagca tgtgagcaaa aggccagcaa
4081 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
4141 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
4201 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
4261 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
4321 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
4381 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
4441 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
4501 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
4561 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
4621 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
4681 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
4741 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
4801 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
4861 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
4921 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
4981 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
5041 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
5101 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
5161 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
5221 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
5281 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
5341 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
5401 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
5461 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
5521 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
5581 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
5641 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
5701 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
5761 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
5821 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
5881 tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat
5941 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
6001 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
6061 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat
6121 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
6181 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
6241 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
6301 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
6361 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
6421 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
6481 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
6541 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
6601 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
6661 gtgtacggtg ggaggtctat ataagcagcg cgttttgcct gtactgggtc tctctggtta
6721 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
6781 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
6841 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca
6901 gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg
6961 aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta
7021 gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta
7081 gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac
7141 atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa
7201 catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag
7261 aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag
7321 agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga
7381 ccaccgcaca gcaagcggcc ggccgctgat cttcagacct ggaggaggag atatgaggga
7441 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc
7501 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc
7561 tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct
7621 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag
7681 ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca
7741 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
7801 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa
7861 atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa
7921 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga
7981 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
8041 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat
8101 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
8161 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg
8221 tggagagaga gacagagaca gatccattcg attagtgaac ggatcggcac tgcgtgcgcc
8281 aattctgcag acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg
8341 ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat
8401 tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc
8461 cagtttggtt agtaccgggc ccgctctaga cgtattaccg ccatgcatta gttattaata
8521 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
8581 tacggtaaat ggcccg
//
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