LOCUS Exported 6975 bp ds-DNA circular SYN 09-JAN-2024 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS AAVS1 hPGK-PuroR-pA donor SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6975) AUTHORS Hockemeyer D, Soldner F, Beard C, Gao Q, Mitalipova M, Dekelver RC, Katibah GE, Amora R, Boydston EA, Zeitler B, Meng X, Miller JC, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jaenisch R TITLE Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. JOURNAL Nat Biotechnol. 2009 Aug 13. ():. PUBMED 19680244 REFERENCE 2 (bases 1 to 6975) AUTHORS . TITLE Direct Submission JOURNAL Exported Jan 9, 2024 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6975 /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" promoter 242..260 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" misc_feature 295..1098 /label=HA-L /note="left homology arm from the adeno-associated virus integration site (AAVS1) within intron 1 of the human PPP1R12C gene" promoter 1111..1611 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 1530..1548 /label=hPGK-F /note="Human PGK promoter, forward primer" CDS 1633..2232 /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(1633..1652) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 2129..2149 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 2239..2463 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" misc_feature 2477..3313 /label=HA-R /note="right homology arm from the adeno-associated virus integration site (AAVS1) within intron 1 of the human PPP1R12C gene" primer_bind complement(3347..3366) /label=T7 /note="T7 promoter, forward primer" promoter complement(3348..3366) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3373..3390) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3373..3389) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3382..3404) /label=M13/pUC Forward /note="In lacZ gene" CDS 3527..3829 /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind 3708..3727 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 4178..4972 /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 GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(4232..4251) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4842..4861 /label=Neo-F /note="Neomycin resistance gene, forward primer" CDS complement(5222..6082) /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 5845..5864 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6206..6794 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6695..6714 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6948..6965 /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 gccaagctca 241 gaattaaccc tcactaaagg gactagtcct gcaggtttaa acgaattcgc cctttgcttt 301 ctctgaccag cattctctcc cctgggcctg tgccgctttc tgtctgcagc ttgtggcctg 361 ggtcacctct acggctggcc cagatccttc cctgccgcct ccttcaggtt ccgtcttcct 421 ccactccctc ttccccttgc tctctgctgt gttgctgccc aaggatgctc tttccggagc 481 acttccttct cggcgctgca ccacgtgatg tcctctgagc ggatcctccc cgtgtctggg 541 tcctctccgg gcatctctcc tccctcaccc aaccccatgc cgtcttcact cgctgggttc 601 ccttttcctt ctccttctgg ggcctgtgcc atctctcgtt tcttaggatg gccttctccg 661 acggatgtct cccttgcgtc ccgcctcccc ttcttgtagg cctgcatcat caccgttttt 721 ctggacaacc ccaaagtacc ccgtctccct ggctttagcc acctctccat cctcttgctt 781 tctttgcctg gacaccccgt tctcctgtgg attcgggtca cctctcactc ctttcatttg 841 ggcagctccc ctacccccct tacctctcta gtctgtgcta gctcttccag ccccctgtca 901 tggcatcttc caggggtccg agagctcagc tagtcttctt cctccaaccc gggcccctat 961 gtccacttca ggacagcatg tttgctgcct ccagggatcc tgtgtccccg agctgggacc 1021 accttatatt cccagggccg gttaatgtgg ctctggttct gggtactttt atctgtcccc 1081 tccaccccac agtggggcaa gcttttgggg ttgcgccttt tccaaggcag ccctgggttt 1141 gcgcagggac gcggctgctc tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg 1201 tctcgcacat tcttcacgtc cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg 1261 ggccccccgg cgacgcttcc tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg 1321 gcgtgccgga cgtgacaaac ggaagccgca cgtctcacta gtaccctcgc agacggacag 1381 cgccagggag caatggcagc gcgccgaccg cgatgggctg tggccaatag cggctgctca 1441 gcagggcgcg ccgagagcag cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg 1501 gtagtgtggg ccctgttcct gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc 1561 acgtcggcag tcggctccct cgttgaccga atcaccgacc tctctcccca gggggatcca 1621 ccggagctta ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc 1681 cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc 1741 gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc 1801 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg 1861 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc 1921 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac 1981 cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc 2041 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg 2101 cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc 2161 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag 2221 cccggtgcct gaggtaccct gtgccttcta gttgccagcc atctgttgtt tgcccctccc 2281 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg 2341 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg 2401 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta 2461 tggggtacca agctttacta gggacaggat tggtgacaga aaagccccat ccttaggcct 2521 cctccttcct agtctcctga tattgggtct aacccccacc tcctgttagg cagattcctt 2581 atctggtgac acacccccat ttcctggagc catctctctc cttgccagaa cctctaaggt 2641 ttgcttacga tggagccaga gaggatcctg ggagggagag cttggcaggg ggtgggaggg 2701 aaggggggga tgcgtgacct gcccggttct cagtggccac cctgcgctac cctctcccag 2761 aacctgagct gctctgacgc ggctgtctgg tgcgtttcac tgatcctggt gctgcagctt 2821 ccttacactt cccaagagga gaagcagttt ggaaaaacaa aatcagaata agttggtcct 2881 gagttctaac tttggctctt cacctttcta gtccccaatt tatattgttc ctccgtgcgt 2941 cagttttacc tgtgagataa ggccagtagc cagccccgtc ctggcagggc tgtggtgagg 3001 aggggggtgt ccgtgtggaa aactcccttt gtgagaatgg tgcgtcctag gtgttcacca 3061 ggtcgtggcc gcctctactc cctttctctt tctccatcct tctttcctta aagagtcccc 3121 agtgctatct gggacatatt cctccgccca gagcagggtc ccgcttccct aaggccctgc 3181 tctgggcttc tgggtttgag tccttggcaa gcccaggaga ggcgctcagg cttccctgtc 3241 ccccttcctc gtccaccatc tcatgcccct ggctctcctg ccccttccct acaggggttc 3301 ctggctctgc tctaagggcg aattcgcggc cgctaaattc aattcgccct atagtgagtc 3361 gtattacaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac 3421 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc 3481 ccgcaccgat cgcccttccc aacagttgcg cagcctatac gtacggcagt ttaaggttta 3541 cacctataaa agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga 3601 cacgccgggg cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt 3661 ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac 3721 cgatatggcc agtgtgccgg tctccgttat cggggaagaa gtggctgatc tcagccaccg 3781 cgaaaatgac atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggcat 3841 gagattatca aaaaggatct tcacctagat ccttttcacg tagaaagcca gtccgcagaa 3901 acggtgctga ccccggatga atgtcagcta ctgggctatc tggacaaggg aaaacgcaag 3961 cgcaaagaga aagcaggtag cttgcagtgg gcttacatgg cgatagctag actgggcggt 4021 tttatggaca gcaagcgaac cggaattgcc agctggggcg ccctctggta aggttgggaa 4081 gccctgcaaa gtaaactgga tggctttctt gccgccaagg atctgatggc gcaggggatc 4141 aagctctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca 4201 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac 4261 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt 4321 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc 4381 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg 4441 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc 4501 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc 4561 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat 4621 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc 4681 cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca 4741 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga 4801 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat 4861 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc 4921 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gaattattaa 4981 cgcttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg 5041 catcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 5101 tacattcaaa tatgtatccg ctcatgagat tatcaaaaag gatcttcacc tagatccttt 5161 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 5221 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 5281 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 5341 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 5401 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 5461 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 5521 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 5581 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 5641 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 5701 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 5761 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 5821 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 5881 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 5941 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 6001 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 6061 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 6121 gttattgtct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 6181 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 6241 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 6301 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 6361 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 6421 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 6481 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 6541 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 6601 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 6661 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 6721 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 6781 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 6841 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 6901 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 6961 gcgaggaagc ggaag //