LOCUS Exported 8102 bp ds-DNA circular SYN 19-JAN-2022 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pLKO.3G SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8102) TITLE pLKO variants REFERENCE 2 (bases 1 to 8102) AUTHORS . TITLE Direct Submission JOURNAL Exported Jan 19, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8102 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 89..110 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 125..155 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 163..179 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 168..190 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 187..203 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 187..203 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 222..242 /label=T3 /note="T3 promoter, forward primer" promoter 224..242 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 270..496 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 497..677 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 724..849 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1342..1575 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1760..1804 /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" promoter 2321..2561 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2321..2341 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2492..2511 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_feature 2661..2778 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2827..3337 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 3256..3274 /label=hPGK-F /note="Human PGK promoter, forward primer" regulatory 3353..3362 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3359..4078 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 3359..4075 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(3404..3425) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(3665..3684) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 4012..4033 /label=EGFP-C /note="EGFP, forward primer" misc_feature 4097..4685 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(4150..4170) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(4568..4579) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(4688..4704) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4689..4705) /label=pBluescriptKS /note="For pBluescript vector" LTR 4890..5123 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 5195..5316 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5232..5251) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5286..5305 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 5356..5491 /label=SV40 ori /note="SV40 origin of replication" primer_bind 5418..5437 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(5511..5530) /label=T7 /note="T7 promoter, forward primer" promoter complement(5512..5530) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(5540..5557) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(5540..5556) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5549..5571) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 5698..6153 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5785..5804) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5995..6016 /label=F1ori-F /note="F1 origin, forward primer" promoter 6179..6283 /gene="bla" /label=AmpR promoter CDS 6284..7144 /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 complement(6502..6521) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7315..7903 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7804..7823 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 8057..8074 /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact 61 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc 121 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat 181 ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa 241 gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct tgtagtcttg 301 caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc accgtgcatg 361 ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac agacgggtct 421 gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat ttaagtgcct 481 agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg agctctctgg 541 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt 601 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt 661 gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg gaaaccagag 721 gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg 781 cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc 841 gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg 901 ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa 961 cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga 1021 cagctacaac catcccttca gacaggatca gaagaactta gatcattata taatacagta 1081 gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac 1141 aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc cgctgatctt 1201 cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt 1261 agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag 1321 agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag 1381 cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat 1441 agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact 1501 cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa 1561 ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca ccactgctgt 1621 gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc acacgacctg 1681 gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct taattgaaga 1741 atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata aatgggcaag 1801 tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat tcataatgat 1861 agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag tgaatagagt 1921 taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga ggggacccga 1981 caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat ccattcgatt 2041 agtgaacgga tctcgacggt atcgatcacg agactagcct cgaccacgac gcaatggaga 2101 aagacggaga gcgccaacgg cgtccatctc gaaggagtcg ccagcgataa ccggagtagt 2161 tgaaatggta ataagacgac caatctgacc agcaaggaag ccaagatggg aaaggtcatg 2221 cggcatacgc tcggcgccag tttgaatatt agacataatt tatcctcaag taagggggta 2281 cacgcgtcag tcgagtcctg tcgacaaggt cgggcaggaa gagggcctat ttcccatgat 2341 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac 2401 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta 2461 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa 2521 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg gtccgcaggt 2581 atgcttgcgt gaattccacc tcgagctact taattaaacc agttttcgac ctcgagacaa 2641 atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg 2701 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt 2761 acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact ttggccgcgg 2821 ctcgaggggg ttggggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg 2881 ctgctctggg cgtggttccg ggaaacgcag cggcgccgac cctgggtctc gcacattctt 2941 cacgtccgtt cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac 3001 gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg 3061 acaaacggaa gccgcacgtc tcactagtac cctcgcagac ggacagcgcc agggagcaat 3121 ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga 3181 gagcagcggc cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct 3241 gttcctgccc gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg 3301 ctccctcgtt gaccgaatca ccgacctctc tccccagggg gatccaccgg tcgccaccat 3361 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg 3421 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg 3481 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct 3541 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca 3601 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt 3661 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt 3721 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa 3781 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg 3841 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga 3901 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta 3961 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct 4021 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaga 4081 tatcaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat 4141 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca 4201 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc 4261 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc 4321 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt 4381 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg 4441 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc 4501 ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta 4561 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg 4621 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc 4681 cccgcatcga taccgtcgac ctcgagacct agaaaaacat ggagcaatca caagtagcaa 4741 tacagcagct accaatgctg attgtgcctg gctagaagca caagaggagg aggaggtggg 4801 ttttccagtc acacctcagg tacctttaag accaatgact tacaaggcag ctgtagatct 4861 tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca 4921 agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct 4981 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca 5041 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta 5101 gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat cttattattc agtatttata 5161 acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg 5221 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc 5281 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg 5341 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat 5401 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc 5461 cagaagtagt gaggaggctt ttttggaggc ctagggacgt acccaattcg ccctatagtg 5521 agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg 5581 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg 5641 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg 5701 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta 5761 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt 5821 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg 5881 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat 5941 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac 6001 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag 6061 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg 6121 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg 6181 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca 6241 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt 6301 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga 6361 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga 6421 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 6481 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca 6541 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt 6601 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac 6661 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct 6721 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 6781 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac 6841 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat 6901 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg 6961 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc 7021 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc 7081 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg 7141 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta 7201 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 7261 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 7321 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 7381 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 7441 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa 7501 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 7561 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 7621 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 7681 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa 7741 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc 7801 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg 7861 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 7921 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc 7981 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag 8041 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa 8101 ac //