LOCUS Exported 6804 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS OCT4-eGFP-2A-Puro SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6804) AUTHORS Hockemeyer D, Wang H, Kiani S, Lai CS, Gao Q, Cassady JP, Cost GJ, Zhang L, Santiago Y, Miller JC, Zeitler B, Cherone JM, Meng X, Hinkley SJ, Rebar EJ, Gregory PD, Urnov FD, Jaenisch R TITLE Genetic engineering of human pluripotent cells using TALE nucleases. JOURNAL Nat Biotechnol. 2011 Jul 7. doi: 10.1038/nbt.1927. PUBMED 21738127 REFERENCE 2 (bases 1 to 6804) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6804 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 107..128 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 143..173 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 181..197 /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 186..208 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 205..221 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 205..221 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" CDS 1008..1724 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 1008..1724 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1053..1074) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1314..1333) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1661..1682 /label=EGFP-C /note="EGFP, forward primer" CDS 1851..2450 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(1851..1870) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 2347..2367 /label=Puro-F /note="Puromycin resistance gene, forward primer" primer_bind complement(3237..3256) /label=T7 /note="T7 promoter, forward primer" promoter complement(3238..3256) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3263..3280) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3263..3279) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3272..3294) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 3420..3848 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3507..3526) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3717..3738 /label=F1ori-F /note="F1 origin, forward primer" CDS 4192..4986 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(4246..4265) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4856..4875 /label=Neo-F /note="Neomycin resistance gene, forward primer" CDS 5004..5864 /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(5222..5241) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6035..6623 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6524..6543 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6777..6794 /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg 241 gtaccgagct cggatccact agtaacggcc gccagtgtgc tggaattcgc ccttttcctg 301 caggtcccac ctgcacagat atgcaaagca gaaaccctcg tgcaggcccg aaagagaaag 361 cgaaccagta tcgagaaccg agtgagaggc aacctggaga atttgttcct gcagtgcccg 421 aaacccacac tgcagcagat cagccacatc gcccagcagc ttgggctcga gaaggatgtg 481 agtgccatgt ctctctgcgg gctccatctc tttcccctgt caccacctcg ctttccctag 541 ctctggctcc tccaactgct ctagggctgt tggctttgga cagaatgtcc aagcagtcag 601 gcctgtctca gctcattctc taatgtcctc ctctaactgc tctagggctg ttggctttgg 661 atagaatgtc caagcagagt caggcccgtc tcagctcatt gtctaatgtc attctccttt 721 ctgtcattca cttgcaggtg gtccgagtgt ggttctgtaa ccggcgccag aagggcaagc 781 gatcaagcag cgactatgca caacgagagg attttgaggc tgctgggtct cctttctcag 841 ggggaccagt gtcctttcct ctggccccag ggccccattt tggtacccca ggctatggga 901 gccctcactt cactgcactg tactcctcgg tccctttccc tgagggggaa gcctttcccc 961 ctgtctccgt caccactctg ggctctccca tgcattcaaa cgctagcatg gtgagcaagg 1021 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg 1081 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc 1141 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc 1201 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct 1261 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg 1321 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg 1381 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca 1441 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga 1501 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc 1561 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc 1621 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg 1681 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtacagc ggtcgcgact 1741 ctagagtcga cggttctggt aagcaaactt tgaattttga ccttcttaag cttgcgggag 1801 acgtcgagtc caaccccggg cccgcatgca agcttcagct gaagcttacc atgaccgagt 1861 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg 1921 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg 1981 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg 2041 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag 2101 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg 2161 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt 2221 tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg 2281 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg 2341 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg 2401 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga ggcgcgccgg 2461 tgcctgccct tctaggaatg ggggacaggg ggaggggagg agctagggaa agaaaacctg 2521 gagtttgtgc cagggttttt gggattaagt tcttcattca ctaaggaagg aattgggaac 2581 acaaagggtg ggggcagggg agtttggggc aactggttgg agggaaggtg aagttcaatg 2641 atgctcttga ttttaatccc acatcatgta tcactttttt cttaaataaa gaagcctggg 2701 acacagtaga tagacacact tatcttggtt tgtccttcag ttactgaggt agggatggga 2761 atatccaatg ctcataccca agtgaccctg aaactaaggt gccatttaca ctccttaagg 2821 tcacacaaca tcagagggag agctgggatt gcagccaagt ttatttgtac agggccctgt 2881 gataggctag ttcccaaaag cctgtgatgc aagaactttt gcccatagac tcagtcacca 2941 tgtagctgtt acctgttcag agctggcttt ttgctttccc accctactct ggaattctta 3001 aatggcttta tacttagaaa tcatcttatt tctgttgaac ctagatcacc ccaaccagaa 3061 acttctatta atactttgtg ctttcttgat accagggtct atttggtttc cacttaaggt 3121 ttttgcatac tctgcccata agtgactcat tagttacggc cggccaaaag ggcgaattct 3181 gcagatatcc atcacactgg cggccgctcg agcatgcatc tagagggccc aattcgccct 3241 atagtgagtc gtattacaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc 3301 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata 3361 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga 3421 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc 3481 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac 3541 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag 3601 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc 3661 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg 3721 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata 3781 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa 3841 cgcgaatttt aacaaaattc agggcgcaag ggctgctaaa ggaagcggaa cacgtagaaa 3901 gccagtccgc agaaacggtg ctgaccccgg atgaatgtca gctactgggc tatctggaca 3961 agggaaaacg caagcgcaaa gagaaagcag gtagcttgca gtgggcttac atggcgatag 4021 ctagactggg cggttttatg gacagcaagc gaaccggaat tgccagctgg ggcgccctct 4081 ggtaaggttg ggaagccctg caaagtaaac tggatggctt tcttgccgcc aaggatctga 4141 tggcgcaggg gatcaagatc tgatcaagag acaggatgag gatcgtttcg catgattgaa 4201 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac 4261 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg 4321 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag 4381 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt 4441 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg 4501 tcatcccacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg 4561 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga 4621 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag 4681 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat 4741 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt 4801 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg 4861 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt 4921 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc 4981 ttctgaattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 5041 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 5101 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 5161 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 5221 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 5281 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 5341 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 5401 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 5461 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 5521 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 5581 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 5641 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 5701 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 5761 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 5821 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 5881 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 5941 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 6001 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 6061 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 6121 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 6181 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 6241 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 6301 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 6361 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 6421 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 6481 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 6541 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 6601 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 6661 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 6721 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 6781 agtcagtgag cgaggaagcg gaag //