LOCUS Exported 7669 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pDIRE SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7669) 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 7669) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7669 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 24..128 /gene="bla" /label=AmpR promoter CDS 129..989 /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(347..366) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1160..1748 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1649..1668 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 1902..1919 /label=L4440 /note="L4440 vector, forward primer" protein_bind 2036..2057 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 2072..2102 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2110..2126 /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 2115..2137 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 2134..2150 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 2134..2150 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 2169..2189 /label=T3 /note="T3 promoter, forward primer" promoter 2171..2189 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind 2217..2233 /label=pBluescriptKS /note="For pBluescript vector" primer_bind 2218..2234 /label=KS primer /note="common sequencing primer, one of multiple similar variants" promoter 2259..2758 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(2294..2317) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" primer_bind 2691..2710 /label=mPGK-F /note="Mouse PGK promoter, forward primer" regulatory 2772..2781 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2778..4076 /codon_start=1 /product="nuclear-targeted site-specific recombinase" /label=FLPo /note="FLPo is a mouse codon-optimized fusion between the SV40 nuclear localization signal and a thermostable version of the Saccharomyces cerevisiae site-specific recombinase FLP. Recombination occurs at FRT sequences." /translation="MAPKKKRKVMSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAA ELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKK LIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESI WEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGV IIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEY QLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWS DKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQL KGSAEGSIRYPAWNGIISQEVLDYLSSYINRRI" primer_bind complement(4104..4121) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4110..4334 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(4428..4447) /label=T7 /note="T7 promoter, forward primer" promoter complement(4429..4447) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4457..4474) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4457..4473) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4466..4488) /label=M13/pUC Forward /note="In lacZ gene" promoter 4800..5978 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 5031..5969 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 5926..5946 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" regulatory 5995..6004 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 6007..6027 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" polyA_signal 7176..7257 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(7213..7232) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 7267..7286 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(7308..7666) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7348..7369) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 7560..7579 /label=F1ori-R /note="F1 origin, reverse primer" ORIGIN 1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 61 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 121 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 241 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 301 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 421 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 481 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 541 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 661 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 721 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 841 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 961 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1021 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1081 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1141 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1201 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1261 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1321 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1381 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1441 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1501 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1561 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1621 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1681 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1741 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1801 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1861 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1921 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1981 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2041 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2101 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2161 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggtaccgggc cccccctcga 2221 ggtcgacggt atcgataagc ttgatatcga attctaccgg gtaggggagg cgcttttccc 2281 aaggcagtct ggagcatgcg ctttagcagc cccgctgggc acttggcgct acacaagtgg 2341 cctctggcct cgcacacatt ccacatccac cggtaggcgc caaccggctc cgttctttgg 2401 tggccccttc gcgccacctt ctactcctcc cctagtcagg aagttccccc ccgccccgca 2461 gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt ctcactagtc tcgtgcagat 2521 ggacagcacc gctgagcaat ggaagcgggt aggcctttgg ggcagcggcc aatagcagct 2581 ttgctccttc gctttctggg ctcagaggct gggaaggggt gggtccgggg gcgggctcag 2641 gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc ggaggcccgg cattctgcac 2701 gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc tcatctccgg gcctttcgac 2761 ctgcaatcgc cgccaccatg gctcctaaga agaagaggaa ggtgatgagc cagttcgaca 2821 tcctgtgcaa gacccccccc aaggtgctgg tgcggcagtt cgtggagaga ttcgagaggc 2881 ccagcggcga gaagatcgcc agctgtgccg ccgagctgac ctacctgtgc tggatgatca 2941 cccacaacgg caccgccatc aagagggcca ccttcatgag ctacaacacc atcatcagca 3001 acagcctgag cttcgacatc gtgaacaaga gcctgcagtt caagtacaag acccagaagg 3061 ccaccatcct ggaggccagc ctgaagaagc tgatccccgc ctgggagttc accatcatcc 3121 cttacaacgg ccagaagcac cagagcgaca tcaccgacat cgtgtccagc ctgcagctgc 3181 agttcgagag cagcgaggag gccgacaagg gcaacagcca cagcaagaag atgctgaagg 3241 ccctgctgtc cgagggcgag agcatctggg agatcaccga gaagatcctg aacagcttcg 3301 agtacaccag caggttcacc aagaccaaga ccctgtacca gttcctgttc ctggccacat 3361 tcatcaactg cggcaggttc agcgacatca agaacgtgga ccccaagagc ttcaagctgg 3421 tgcagaacaa gtacctgggc gtgatcattc agtgcctggt gaccgagacc aagacaagcg 3481 tgtccaggca catctacttt ttcagcgcca gaggcaggat cgaccccctg gtgtacctgg 3541 acgagttcct gaggaacagc gagcccgtgc tgaagagagt gaacaggacc ggcaacagca 3601 gcagcaacaa gcaggagtac cagctgctga aggacaacct ggtgcgcagc tacaacaagg 3661 ccctgaagaa gaacgccccc taccccatct tcgctatcaa gaacggccct aagagccaca 3721 tcggcaggca cctgatgacc agctttctga gcatgaaggg cctgaccgag ctgacaaacg 3781 tggtgggcaa ctggagcgac aagagggcct ccgccgtggc caggaccacc tacacccacc 3841 agatcaccgc catccccgac cactacttcg ccctggtgtc caggtactac gcctacgacc 3901 ccatcagcaa ggagatgatc gccctgaagg acgagaccaa ccccatcgag gagtggcagc 3961 acatcgagca gctgaagggc agcgccgagg gcagcatcag ataccccgcc tggaacggca 4021 tcatcagcca ggaggtgctg gactacctga gcagctacat caacaggcgg atctgatgag 4081 atatcctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt 4141 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta 4201 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg 4261 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc 4321 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctcgagat ccactagttc 4381 tagcctcgag gctagagcgg ccgccaccgc ggtggagctc caattcgccc tatagtgagt 4441 cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 4501 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag 4561 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggaaattgt 4621 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa 4681 ccaataggcc gaaatcggca aaatccctta atcaagcttt gcaaagatgg ataaagtttt 4741 aaacagagag gaatctttgc agctaatgga ccttctaggt cttgaaagga gtgggaattg 4801 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg 4861 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg 4921 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag 4981 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg 5041 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta 5101 cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg 5161 agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc 5221 ttgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt 5281 tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc 5341 aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg 5401 cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag 5461 cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg 5521 gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag 5581 ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga 5641 cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt 5701 cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt 5761 agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg 5821 agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat 5881 tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag 5941 tggttcaaag tttttttctt ccatttcagg tgtcgtgaag gaattggatc cgccgccacc 6001 atggtgccca agaagaagag gaaagtctcc aacctgctga ctgtgcacca aaacctgcct 6061 gccctccctg tggatgccac ctctgatgaa gtcaggaaga acctgatgga catgttcagg 6121 gacaggcagg ccttctctga acacacctgg aagatgctcc tgtctgtgtg cagatcctgg 6181 gctgcctggt gcaagctgaa caacaggaaa tggttccctg ctgaacctga ggatgtgagg 6241 gactacctcc tgtacctgca agccagaggc ctggctgtga agaccatcca acagcacctg 6301 ggccagctca acatgctgca caggagatct ggcctgcctc gcccttctga ctccaatgct 6361 gtgtccctgg tgatgaggag aatcagaaag gagaatgtgg atgctgggga gagagccaag 6421 caggccctgg cctttgaacg cactgacttt gaccaagtca gatccctgat ggagaactct 6481 gacagatgcc aggacatcag gaacctggcc ttcctgggca ttgcctacaa caccctgctg 6541 cgcattgccg aaattgccag aatcagagtg aaggacatct cccgcaccga tggtgggaga 6601 atgctgatcc acattggcag gaccaagacc ctggtgtcca cagctggtgt ggagaaggcc 6661 ctgtccctgg gggttaccaa gctggtggag agatggatct ctgtgtctgg tgtggctgat 6721 gaccccaaca actacctgtt ctgccgggtc agaaagaatg gtgtggctgc cccttctgcc 6781 acctcccaac tgtccacccg ggccctggaa gggatctttg aggccaccca ccgcctgatc 6841 tatggtgcca aggatgactc tgggcagaga tacctggcct ggtctggcca ctctgccaga 6901 gtgggtgctg ccagggacat ggccagggct ggtgtgtcca tccctgaaat catgcaggct 6961 ggtggctgga ccaatgtgaa cattgtgatg aactacatca gaaacctgga ctctgagact 7021 ggggccatgg tgaggctgct cgaggatggg gactgaacta gtgactctag atcataatca 7081 gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga 7141 acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg 7201 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt 7261 ctagttgtgg tttgtccaaa ctcatcaatg tatactagta tagagattaa atcaaaagaa 7321 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac 7381 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa 7441 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct 7501 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa 7561 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc 7621 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcag //