LOCUS Exported 7954 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS PITX3-eGFP-PGK-Puro SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7954) 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 7954) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7954 /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 1011..1730 /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 1011..1727 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1056..1077) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1317..1336) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1664..1685 /label=EGFP-C /note="EGFP, forward primer" protein_bind complement(1761..1794) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 1855..2351 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(1890..1913) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" primer_bind 2284..2303 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 2391..2990 /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(2391..2410) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 2887..2907 /label=Puro-F /note="Puromycin resistance gene, forward primer" protein_bind complement(3547..3580) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(4387..4406) /label=T7 /note="T7 promoter, forward primer" promoter complement(4388..4406) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4413..4430) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4413..4429) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4422..4444) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4570..4998 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4657..4676) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4867..4888 /label=F1ori-F /note="F1 origin, forward primer" CDS 5342..6136 /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(5396..5415) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 6006..6025 /label=Neo-F /note="Neomycin resistance gene, forward primer" CDS 6154..7014 /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(6372..6391) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7185..7773 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7674..7693 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7927..7944 /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 caggcctcat ctcgtttatt gaccgcaggg ccccggctgg aagccccgtc ggggtggccc 361 gcccttcagc cgctgggacc cggccccggg ccctgaccgc ctttctcccg tgcccgcagg 421 tgtggttcaa gaaccggcgc gccaaatggc ggaagcgcga gcgcagccag caggccgagc 481 tatgcaaagg cagcttcgcg gcgccgctcg gggggctggt gccgccctac gaggaggtgt 541 accccggcta ctcgtacggc aactggccgc ccaaggctct tgccccgccg ctcgccgcca 601 agacctttcc attcgccttc aactcggtca acgtggggcc tctggcttcg cagcccgtct 661 tctcgccacc cagctccatc gccgcctcca tggtgccctc cgccgcggct gccccgggca 721 ccgtgccagg gcctggggcc ctgcagggcc tgggcggggg cccccccggg ctggctccgg 781 ccgccgtgtc ctccggggcc gtgtcctgcc cttatgcctc ggccgccgcc gccgccgcgg 841 ctgccgcctc ttccccctac gtctatcggg acccgtgtaa ctcgagcctg gccagcctgc 901 ggctcaaagc caaacagcac gcctccttca gctaccccgc tgtgcacggg ccgcccccgg 961 cagccaacct tagtccgtgc cagtacgccg tggaaaggcc cgtagctagc atggtgagca 1021 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa 1081 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga 1141 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca 1201 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact 1261 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg 1321 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca 1381 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt 1441 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg 1501 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc 1561 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca 1621 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt 1681 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa gaattccgat 1741 catattcaat aacccttaat ataacttcgt ataatgtatg ctatacgaag ttattaggtc 1801 tgaagaggag tttacgtcca gccaagctta ggatctcgac ctcgaaattc taccgggtag 1861 gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg ctggcacttg 1921 gcgctacaca agtggcctct ggcctcgcac acattccaca tccaccggta gcgccaaccg 1981 gctccgttct ttggtggccc cttcgcgcca ccttctactc ctcccctagt caggaagttc 2041 ccccccgccc cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact 2101 agtctcgtgc agatggacag caccgctgag caatggaagc gggtaggcct ttggggcagc 2161 ggccaatagc agctttgctc cttcgctttc tgggctcagc agctgggaag ggtgggtccg 2221 ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc 2281 cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc 2341 cgggcctttc gacctgcatc catctagatc tcgagcagct gaagcttacc atgaccgagt 2401 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg 2461 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg 2521 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg 2581 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag 2641 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg 2701 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt 2761 tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg 2821 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg 2881 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg 2941 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc 3001 acgacccgca gcgcccgacc gaaaggagcg cacgacccca tgcatcgatg atatcagatc 3061 cccgggatgc agaaattgat gatctattaa acaataaaga tgtccactaa aatggaagtt 3121 tttcctgtca tactttgtta agaagggtga gaacagagta cctacatttt gaatggaagg 3181 attggagcta cgggggtggg ggtggggtgg gattagataa atgcctgctc tttactgaag 3241 gctctttact attgctttat gataatgttt catagttgga tatcataatt taaacaagca 3301 aaaccaaatt aagggccagc tcattcctcc cactcatgat ctatagatct atagatctct 3361 cgtgggatca ttgtttttct cttgattccc actttgtggt tctaagtact gtggtttcca 3421 aatgtgtcag tttcatagcc tgaagaacga gatcagcagc ctctgttcca catacacttc 3481 attctcagta ttgttttgcc aagttctaat tccatcagaa gctggtcgag atccggaacc 3541 cttaatataa cttcgtataa tgtatgctat acgaagttat taggtccctc gaagaggttc 3601 actaggcgcg ccgcggcccc gcccgtagat catccccgag ggcgggggca acgattcaca 3661 gcctccgcgg actggggtca ttttgactgg cttgctcccg ccccagggtc tgaaaggggt 3721 gtttgggcag ctggggggca ccggctcagg agagggcctt cccctcccag ccctgagggg 3781 tggactaggc cctacacaca gaccgcgccc ctgggactaa agccaggaac agggaccagc 3841 tccccggggg ccaactcacc cttggcccat cccgccttct ccaggcttcc cctccctcgt 3901 tttcaaagat aaatgaaata aacgtgcgcg gactgtcgaa ggcgtgatga ttcaaagttg 3961 cgcttgagtc ctcttccctt catcctcccc cactgtctac aggcgagctg gatgtacagg 4021 aacgaagtga gggtccaggc ttctagggac tcagtcccac tcaccacccc ccctactcca 4081 ggtcggggca gcccgggatt cctgcagcaa agaggtgcgt gcgtgcgggg gacgtgggaa 4141 accggctacc tggctggcat cggccacagc cggtgctgcg gcgccacctc gtggtctcag 4201 ggaggctagc tggtagatct ggtctccaac gcttccttgt aggtgtcagc tctgctattc 4261 agggagccta tgactttaga taaagttact ccgcctttct gcacctgggc cggccaaaag 4321 ggcgaattct gcagatatcc atcacactgg cggccgctcg agcatgcatc tagagggccc 4381 aattcgccct atagtgagtc gtattacaat tcactggccg tcgttttaca acgtcgtgac 4441 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc 4501 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat 4561 ggcgaatgga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 4621 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 4681 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 4741 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac 4801 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct 4861 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt 4921 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac 4981 aaaaatttaa cgcgaatttt aacaaaattc agggcgcaag ggctgctaaa ggaagcggaa 5041 cacgtagaaa gccagtccgc agaaacggtg ctgaccccgg atgaatgtca gctactgggc 5101 tatctggaca agggaaaacg caagcgcaaa gagaaagcag gtagcttgca gtgggcttac 5161 atggcgatag ctagactggg cggttttatg gacagcaagc gaaccggaat tgccagctgg 5221 ggcgccctct ggtaaggttg ggaagccctg caaagtaaac tggatggctt tcttgccgcc 5281 aaggatctga tggcgcaggg gatcaagatc tgatcaagag acaggatgag gatcgtttcg 5341 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt 5401 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc 5461 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact 5521 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt 5581 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca 5641 ggatctcctg tcatcccacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat 5701 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg 5761 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga 5821 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga 5881 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa 5941 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga 6001 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt 6061 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct 6121 tgacgagttc ttctgaattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 6181 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 6241 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 6301 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 6361 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 6421 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 6481 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 6541 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 6601 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 6661 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 6721 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 6781 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 6841 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 6901 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 6961 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 7021 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 7081 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 7141 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 7201 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 7261 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 7321 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 7381 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 7441 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 7501 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 7561 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 7621 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 7681 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 7741 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 7801 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 7861 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 7921 gagcgcagcg agtcagtgag cgaggaagcg gaag //