LOCUS Exported 5232 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION PuroR containing plasmid for the generation of gene disruptions via Bxb1-mediated recombination into MIN-tagged cell lines. This vector can also be used to express cDNAs.. ACCESSION . VERSION . KEYWORDS attB‐mCh­‐Poly(A)-­PuroR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5232) AUTHORS Mulholland CB, Smets M, Schmidtmann E, Leidescher S, Markaki Y, Hofweber M, Qin W, Manzo M, Kremmer E, Thanisch K, Bauer C, Rombaut P, Herzog F, Leonhardt H, Bultmann S TITLE A modular open platform for systematic functional studies under physiological conditions. JOURNAL Nucleic Acids Res. 2015 May 24. pii: gkv550. PUBMED 26007658 REFERENCE 2 (bases 1 to 5232) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5232 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 62..561 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(97..120) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" primer_bind 494..513 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 585..1181 /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(585..604) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 1081..1101 /label=Puro-F /note="Puromycin resistance gene, forward primer" CDS 1188..2183 /codon_start=1 /product="truncated herpes simplex virus thymidine kinase (Salomon et al., 1995)" /label=-Delta-TK /note="confers sensitivity to nucleoside analogs such as acyclovir or ganciclovir" /translation="MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWRV LGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGS SHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGAL PEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQCGGSWREDWGQLSGTAV PPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRSMHVFI LDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN" primer_bind complement(2236..2253) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2242..2466 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" CDS 2535..3239 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE GRHSTGGMDELYK" primer_bind complement(2677..2695) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 2945..2964 /label=mCherry-F /note="mCherry, forward primer" regulatory 3285..3294 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" polyA_signal 3347..3481 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3371..3390) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 3425..3444 /label=SV40pA-R /note="SV40 polyA, reverse primer" promoter 3617..3664 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 3683..4486 /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3746..3765) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4356..4375 /label=Neo-F /note="Neomycin resistance gene, forward primer" rep_origin complement(4615..5203) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4695..4714) /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 gatgccagct cattcctccc actcatgatc tatagatccc ccgggctgca ggaattctac 61 cgggtagggg aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg 121 ggcacttggc gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg 181 cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc 241 aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca 301 cgtctcacta gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt 361 tggggcagcg gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg 421 ggtgggtccg ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc 481 tccggaggcc cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct 541 tcctcatctc cgggcctttc gaccgatcat caagcttgat cctcatgacc gagtacaagc 601 ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg 661 cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg 721 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg 781 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg 841 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc 901 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg 961 ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc 1021 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc 1081 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg 1141 aaggaccgcg cacctggtgc atgacccgca agcccggtgc cggatccatg cccacgctac 1201 tgcgggttta tatagacggt ccccacggga tggggaaaac caccaccacg caactgctgg 1261 tggccctggg ttcgcgcgac gatatcgtct acgtacccga gccgatgact tactggcggg 1321 tgctgggggc ttccgagaca atcgcgaaca tctacaccac acaacaccgc ctcgaccagg 1381 gtgagatatc ggccggggac gcggcggtgg taatgacaag cgcccagata acaatgggca 1441 tgccttatgc cgtgaccgac gccgttctgg ctcctcatat cgggggggag gctgggagct 1501 cacatgcccc gcccccggcc ctcaccctca tcttcgaccg ccatcccatc gccgccctcc 1561 tgtgctaccc ggccgcgcgg taccttatgg gcagcatgac cccccaggcc gtgctggcgt 1621 tcgtggccct catcccgccg accttgcccg gcaccaacat cgtgcttggg gcccttccgg 1681 aggacagaca catcgaccgc ctggccaaac gccagcgccc cggcgagcgg ctggacctgg 1741 ctatgctggc tgcgattcgc cgcgtttacg ggctacttgc caatacggtg cggtatctgc 1801 agtgcggcgg gtcgtggcgg gaggactggg gacagctttc ggggacggcc gtgccgcccc 1861 agggtgccga gccccagagc aacgcgggcc cacgacccca tatcggggac acgttattta 1921 ccctgtttcg ggcccccgag ttgctggccc ccaacggcga cctgtataac gtgtttgcct 1981 gggccttgga cgtcttggcc aaacgcctcc gttccatgca cgtctttatc ctggattacg 2041 accaatcgcc cgccggctgc cgggacgccc tgctgcaact tacctccggg atggtccaga 2101 cccacgtcac cacccccggc tccataccga cgatatgcga cctggcgcgc acgtttgccc 2161 gggagatggg ggaggctaac tgagctctag agcggccagt gtcgcggtat cgatgatcta 2221 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct 2281 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg 2341 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc 2401 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct 2461 ctatggcttc tgaggcggaa agaaccagca tcccggcttg tcgacgacgg cggtctccgt 2521 cgtcaggatc atccgtgagc aagggcgagg aggataacat ggccatcatc aaggagttca 2581 tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg 2641 agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg 2701 gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct 2761 acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca 2821 agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct 2881 ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg 2941 acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc 3001 ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc 3061 actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg 3121 cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg 3181 aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaaga 3241 gcggcctgag gagcagagcc caggcgagca acagcgccgt ggacgccacc atgggcgatc 3301 gcccgggaat tgactagtgc ggccgcagtt aacgaattct ctagaggatc cagacatgat 3361 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat 3421 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt 3481 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt 3541 ttaaagcaag taaaacctct acaaatgtgg tatggctgat tatgatcctg caagcctcgt 3601 cgccgcggtt tattctgttg acaattaatc atcggcatag tatatcggca tagtataata 3661 cgacaaggtg aggaactaaa ccatgggatc ggccattgaa caagatggat tgcacgcagg 3721 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacgatcgg 3781 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa 3841 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct 3901 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga 3961 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc 4021 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac 4081 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc 4141 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact 4201 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga 4261 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg 4321 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga 4381 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgcccccga 4441 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg 4501 ttcgaataaa gaccgaccaa gcgacgtctg agagctccct ggcgaattcg gtaccaataa 4561 aagagcttta ttttcatgat ctgtgtgttg gtttttggcc gcgttgctgg cgtttttcca 4621 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 4681 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 4741 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 4801 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 4861 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 4921 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 4981 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 5041 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 5101 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 5161 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 5221 ttctagtgtg cg //