LOCUS Exported 6318 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pDREV-0 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6318) AUTHORS Osterwalder M, Galli A, Rosen B, Skarnes WC, Zeller R, Lopez-Rios J. TITLE Dual RMCE for efficient re-engineering of mouse mutant alleles JOURNAL Nat Methods. 2010 Oct 17 PUBMED 20953177 REFERENCE 2 (bases 1 to 6318) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6318 /organism="synthetic DNA construct" /mol_type="other DNA" terminator 103..189 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 281..308 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" primer_bind 316..339 /label=pENTR-F /note="pENTR vectors, forward primer" protein_bind 358..457 /gene="mutant version of attL" /label=attL1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" protein_bind 470..517 /label=FRT /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011)." misc_feature 1309..1424 /label=En2 SA /note="splice acceptor from the mouse Engrailed 2 gene (Skarnes et al., 1992)" CDS 1435..1488 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 1495..1866 /codon_start=1 /gene="HIST1H2BJ" /product="human histone H2B" /label=H2B /translation="PEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKVL KQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPG ELAKHAVSEGTKAVTKYTSA" CDS 1876..2595 /codon_start=1 /product="yellow fluorescent protein (YFP) with fast and efficient maturation (Nagai et al., 2002)" /label=Venus /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1921..1942) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2182..2201) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2529..2550 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 2718..2852 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2755..2774) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2809..2828 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" promoter 2935..3434 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(2970..2993) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" primer_bind 3367..3386 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 3474..4073 /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(3474..3493) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 3970..3990 /label=Puro-F /note="Puromycin resistance gene, forward primer" protein_bind 4654..4687 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind complement(4709..4808) /gene="mutant version of attL" /label=attL2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 4907..4954 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 4973..5545 /codon_start=1 /gene="Streptomyces noursei nat1" /product="nourseothricin acetyltransferase" /label=NrsR /note="confers resistance to nourseothricin" /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT LCGLDTALYDGTASDGEQALYMSMPCP" rep_origin 5668..6256 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6157..6176 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgctagca tggatctcgg 61 ggacgtctaa ctactaagcg agagtaggga actgccaggc atcaaataaa acgaaaggct 121 cagtcggaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt 181 aggacaaatc cgccgggagc ggatttgaac gttgtgaagc aacggcccgg agggtggcgg 241 gcaggacgcc cgccataaac tgccaggcat caaactaagc agaaggccat cctgacggat 301 ggcctttttg cgtttctaca aactcttcct gttagttagt tacttaagct cgggccccaa 361 ataatgattt tattttgact gatagtgacc tgttcgttgc aacaaattga taagcaatgc 421 ttttttataa tgccaacttt gtacaaaaaa gcaggctggc gccggaaccg aagttcctat 481 tccgaagttc ctattctcta gaaagtatag gaacttcgaa ccctttccca caccaccctc 541 cacacttgcc ccaaacactg ccaactatgt aggaggaagg ggttgggact aacagaagaa 601 cccgttgtgg ggaagctgtt gggagggtca ctttatgttc ttgcccaagg tcagttgggt 661 ggcctgcttc tgatgaggtg gtcccaaggt ctggggtaga aggtgagagg gacaggccac 721 caaggtcagc cccccccccc tatcccatag gagccaggtc cctctcctgg acaggaagac 781 tgaaggggag atgccagaga ctcagtgaag cctggggtac cctattggag tccttcaagg 841 aaacaaactt ggcctcacca ggcctcagcc ttggctcctc ctgggaactc tactgccctt 901 gggatcccct tgtagttgtg ggttacatag gaagggggac gggattcccc ttgactggct 961 agcctactct tttcttcagt cttctccatc tcctctcacc tgtctctcga ccctttccct 1021 aggatagact tggaaaaaga taaggggaga aaacaaatgc aaacgaggcc agaaagattt 1081 tggctgggca ttccttccgc tagcttttat tgggatcccc tagtttgtga taggcctttt 1141 agctacatct gccaatccat ctcattttca cacacacaca caccactttc cttctggtca 1201 gtgggcacat gtccagcctc aagtttatat caccaccccc aatgcccaac acttgtatgg 1261 ccttgggcgg gtcatccccc cccccacccc cagtatctgc aacctcaagc tagcttgggt 1321 gcgttggttg tggataagta gctagactcc agcaaccagt aacctctgcc ctttctcctc 1381 catgacaacc aggtcccagg tcccgaaaac caaagaagaa gaacgcagat ctccgagggc 1441 agaggaagtc ttctaacatg cggtgacgtg gaggagaatc ccggaccggg gggaccagag 1501 ccagcgaagt ctgctcccgc cccgaaaaag ggctccaaga aggcggtgac taaggcgcag 1561 aagaaagacg gcaagaagcg caagcgcagc cgcaaggaga gctattccat ctatgtgtac 1621 aaggttctga agcaggtcca ccctgacacc ggcatttcgt ccaaggccat gggcatcatg 1681 aattcgtttg tgaacgacat tttcgagcgc atcgcaggtg aggcttcccg cctggcgcat 1741 tacaacaagc gctcgaccat cacctccagg gagatccaga cggccgtgcg cctgctgctg 1801 cctggggagt tggccaagca cgccgtgtcc gagggtacta aggccgtcac caagtacacc 1861 agcgctaaat ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 1921 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag 1981 ggcgatgcca cctacggcaa gctgaccctg aagctgatct gcaccaccgg caagctgccc 2041 gtgccctggc ccaccctcgt gaccaccctg ggctacggcc tgcagtgctt cgcccgctac 2101 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag 2161 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc 2221 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 2281 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcaccgcc 2341 gacaagcaga agaacggcat caaggccaac ttcaagatcc gccacaacat cgaggacggc 2401 ggcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg 2461 ctgcccgaca accactacct gagctaccag tccgccctga gcaaagaccc caacgagaag 2521 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac 2581 gagctgtaca agtaagaaga gcttaattaa tgatcataat cagccatatc acatctgtag 2641 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga 2701 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata 2761 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca 2821 aactcatcaa tgtatcttat catgtctgga tccgtttaaa ctaactttaa ataattggca 2881 ttatttaaag ttacatatgg cggccgccct aaggctcgac ctcgaaattc taccgggtag 2941 gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg ctgggcactt 3001 ggcgctacac aagtggcctc tggcctcgca cacattccac atccaccggt aggcgccaac 3061 cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta gtcaggaagt 3121 tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca 3181 ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc ctttggggca 3241 gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga aggggtgggt 3301 ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcccgaagg tcttccggag 3361 gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc tcttcctcat 3421 ctccgggcct ttcgacctgc atccatctag atctcgagca gctgaagctt accatgaccg 3481 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc 3541 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatcca gatcgccaca 3601 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca 3661 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg 3721 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc 3781 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt 3841 ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg 3901 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct 3961 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg 4021 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgacgcccgc 4081 cccacgaccc gcagcgcccg accgaaagga gcgcacgacc ccatgcatcg atgatatcag 4141 atccccggga tgcagaaatt gatgatctat taaacaataa agatgtccac taaaatggaa 4201 gtttttcctg tcatactttg ttaagaaggg tgagaacaga gtacctacat tttgaatgga 4261 aggattggag ctacgggggt gggggtgggg tgggattaga taaatgcntg ctctttactg 4321 aaggctcttt actattgctt tatgataatg tttcatagtt ggatatcata atttaaacaa 4381 gcaaaaccaa attaagggcc agctcattcc tcccactcat gatctataga tctatagatc 4441 tctcgtggga tcattgtttt tctcttgatt cccactttgt ggttctaagt actgtggttt 4501 ccaaatgtgt cagtttcata gcctgaagaa cgagatcagc agcctctgtt ccacatacac 4561 ttcattctca gtattgtttt gccaagttct aattccatca gaagctggtc gagtaacttt 4621 aaataattgg cattatttaa agttaggcgc gccataactt cgtatagcat acattatacg 4681 aagttatgtc gagatattta aatattagac ccagctttct tgtacaaagt tggcattata 4741 agaaagcatt gcttatcaat ttgttgcaac gaacaggtca ctatcagtca aaataaaatc 4801 attatttgcc atccagctgc agctctggcc cgtgtctcaa aatctctgat gttacattgc 4861 acaagataaa agcttgtata tccattttcg gatctgatca gcacgtgttg acaattaatc 4921 atcggcatag tatatcggca tagtataata cgacaaggtg aggaactaaa ccatgggtac 4981 cactcttgac gacacggctt accggtaccg caccagtgtc ccgggggacg ccgaggccat 5041 cgaggcactg gatgggtcct tcactaccga caccgtcttc cgcgtcaccg ccaccgggga 5101 cggcttcacc ctgcgggagg tgccggtgga cccgcccctg accaaggtgt tccccgacga 5161 cgaatcggac gacgaatcgg acgacgggga ggacggtgac ccggactctc ggacgttcgt 5221 cgcgtacggg gacgacggcg acctggcggg cttcgtggtc gtctcgtact ccggctggaa 5281 ccgccggctg accgtcgagg acatcgaggt cgccccggag caccgggggc acggggtcgg 5341 gcgcgcgttg atggggctcg cgacggagtt cgcccgcgag cggggtgccg ggcacctctg 5401 gctggaggtc accaacgtca acgcaccggc gatccacgcg taccggcgga tggggttcac 5461 cctctgcggc ctggacaccg ccctgtacga cggcaccgcc tcggacggcg agcaggcgct 5521 ctacatgagc atgccctgcc cctaagcact tcgtggccga ggagcaggac tgacacgttc 5581 agaattggtt aattggttgt aacattattc agattgggcc ccgttccact gagcgtcaga 5641 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 5701 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 5761 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct 5821 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 5881 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 5941 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 6001 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 6061 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 6121 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 6181 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 6241 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 6301 gccttttgct cacatgtt //