LOCUS Exported 7982 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION episomal expression of FlpO and Cre recombinases. ACCESSION . VERSION . KEYWORDS pCag FlpO-2A-Cre EV SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7982) AUTHORS Kim GB, Rincon Fernandez Pacheco D, Saxon D, Yang A, Sabet S, Dutra-Clarke M, Levy R, Watkins A, Park H, Abbasi Akhtar A, Linesch PW, Kobritz N, Chandra SS, Grausam K, Ayala-Sarmiento A, Molina J, Sedivakova K, Hoang K, Tsyporin J, Gareau DS, Filbin MG, Bannykh S, Santiskulvong C, Wang Y, Tang J, Suva ML, Chen B, Danielpour M, Breunig JJ TITLE Rapid Generation of Somatic Mouse Mosaics with Locus-Specific, Stably Integrated Transgenic Elements. JOURNAL Cell. 2019 Sep 19;179(1):251-267.e24. doi: 10.1016/j.cell.2019.08.013. PUBMED 31539496 REFERENCE 2 (bases 1 to 7982) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7982 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 50..429 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" intron 707..1723 /label=chimeric intron /note="chimera between introns from chicken beta-actin and rabbit beta-globin" primer_bind 1647..1669 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 1731..1750 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" promoter 1770..1787 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" intron 1836..2065 /label=chimeric intron /note="chimera between introns from adenovirus and immunoglobulin heavy chain genes" primer_bind 2048..2067 /label=pMT2-F /note="Synthetic intron, forward primer" CDS 3386..3439 /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 3440..4468 /codon_start=1 /product="site-specific recombinase" /label=Cre /note="Cre is a site-specific recombinase from bacteriophage P1. Recombination occurs at loxP sequences." /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED GD" polyA_signal 4611..4732 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4622..4641) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 4676..4695 /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind complement(4947..4967) /label=T3 /note="T3 promoter, forward primer" promoter complement(4947..4965) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(4986..5002) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4986..5002) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4999..5021) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5010..5026 /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)." promoter complement(5034..5064) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5079..5100 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5217..5234) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5388..5976) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5468..5487) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6147..7007) /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 6770..6789 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7008..7112) /gene="bla" /label=AmpR promoter rep_origin complement(7138..7593) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7275..7296) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 7487..7506 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 7720..7742 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 7734..7751 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 7735..7751 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 7761..7780 /label=T7 /note="T7 promoter, forward primer" promoter 7761..7779 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" ORIGIN 1 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta 61 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag 121 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc 181 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga 241 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat 301 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc 361 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct 421 attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc 481 ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg 541 gggggggggg ggcgcgccrg gsggggsggg gsggggsgrg gggsggggsg gggsgrggcg 601 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag 661 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg 721 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact 781 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta 841 gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct 901 ccgggagggc cctttgtgcg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt 961 ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc 1021 ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg 1081 gtgcgggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg tgggggggtg 1141 agcagggggt gtgggcgcgt cggtcgggct gcaacccccc ctgcaccccc ctccccgagt 1201 tgctgagcac ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg cggggctcgc 1261 cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc 1321 cggggrgggc tcgggggagg ggcgcggcgg cccccggagc gccggcggct gtcgaggcgc 1381 ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg gacttccttt 1441 gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg 1501 cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc ttcgtgcgtc 1561 gccgcgccgc cgtccccttc tccctctcca gcctcggggc tgtccgcggg gggacggctg 1621 ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag 1681 agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct 1741 ggttattgtg ctgtctcatc attttggcaa atacgactca ctatagggag acccaagctt 1801 ctagaattcg ctgtctgcga gggccagctg ttggggtgag tactccctct caaaagcggg 1861 catgacttct gcgctaagat tgtcagtttc caaaaacgag gaggatttga tattcacctg 1921 gcccgcggtg atgcctttga gggtggccgc gtccatctgg tcagaaaaga caatcttttt 1981 gttgtcaagc ttgaggtgtg gcaggcttga gatctggcca tacacttgag tgacaatgac 2041 atccactttg cctttctctc cacaggtgtc cactcccagg tccaactgca gcccaagcta 2101 gaccatgagc cagttcgaca tcctgtgcaa gacccccccc aaggtgctgg tgcggcagtt 2161 cgtggagaga ttcgagaggc ccagcggcga gaagatcgcc agctgtgccg ccgagctgac 2221 ctacctgtgc tggatgatca cccacaacgg caccgccatc aagagggcca ccttcatgag 2281 ctacaacacc atcatcagca acagcctgag cttcgacatc gtgaacaaga gcctgcagtt 2341 caagtacaag acccagaagg ccaccatcct ggaggccagc ctgaagaagc tgatccccgc 2401 ctgggagttc accatcatcc cttacaacgg ccagaagcac cagagcgaca tcaccgacat 2461 cgtgtccagc ctgcagctgc agttcgagag cagcgaggag gccgacaagg gcaacagcca 2521 cagcaagaag atgctgaagg ccctgctgtc cgagggcgag agcatctggg agatcaccga 2581 gaagatcctg aacagcttcg agtacaccag caggttcacc aagaccaaga ccctgtacca 2641 gttcctgttc ctggccacat tcatcaactg cggcaggttc agcgacatca agaacgtgga 2701 ccccaagagc ttcaagctgg tgcagaacaa gtacctgggc gtgatcattc agtgcctggt 2761 gaccgagacc aagacaagcg tgtccaggca catctacttt ttcagcgcca gaggcaggat 2821 cgaccccctg gtgtacctgg acgagttcct gaggaacagc gagcccgtgc tgaagagagt 2881 gaacaggacc ggcaacagca gcagcaacaa gcaggagtac cagctgctga aggacaacct 2941 ggtgcgcagc tacaacaagg ccctgaagaa gaacgccccc taccccatct tcgctatcaa 3001 gaacggccct aagagccaca tcggcaggca cctgatgacc agctttctga gcatgaaggg 3061 cctgaccgag ctgacaaacg tggtgggcaa ctggagcgac aagagggcct ccgccgtggc 3121 caggaccacc tacacccacc agatcaccgc catccccgac cactacttcg ccctggtgtc 3181 caggtactac gcctacgacc ccatcagcaa ggagatgatc gccctgaagg acgagaccaa 3241 ccccatcgag gagtggcagc acatcgagca gctgaagggc agcgccgagg gcagcatcag 3301 ataccccgcc tggaacggca tcatcagcca ggaggtgctg gactacctga gcagctacat 3361 caacaggcgg atctctagaa gagccgaggg caggggaagt cttctaacat gcggggacgt 3421 ggaggaaaat cccgggccct ccaatttact gaccgtacac caaaatttgc ctgcattacc 3481 ggtcgatgca acgagtgatg aggttcgcaa gaacctgatg gacatgttca gggatcgcca 3541 ggcgttttct gagcatacct ggaaaatgct tctgtccgtt tgccggtcgt gggcggcatg 3601 gtgcaagttg aataaccgga aatggtttcc cgcagaacct gaagatgttc gcgattatct 3661 tctatatctt caggcgcgcg gtctggcagt aaaaactatc cagcaacatt tgggccagct 3721 aaacatgctt catcgtcggt ccgggctgcc acgaccaagt gacagcaatg ctgtttcact 3781 ggttatgcgg cgcattcgaa aagaaaacgt tgatgccggt gaacgtgcaa aacaggctct 3841 agcgttcgaa cgcactgatt tcgaccaggt tcgttcactc atggaaaata gcgatcgctg 3901 ccaggatata cgtaatctgg catttctggg gattgcttat aacaccctgt tacgtatagc 3961 cgaaattgcc aggatcaggg ttaaagatat ctcacgtact gacggtggga gaatgttaat 4021 ccatattggc agaacgaaaa cgctggttag caccgcaggt gtagagaagg cacttagcct 4081 gggggtaact aaactggtcg agcgatggat ttccgtctct ggtgtagctg atgatccgaa 4141 taactacctg ttttgccggg tcagaaaaaa tggtgttgcc gcgccatctg ccaccagcca 4201 gctatcaact cgcgccctgg aagggatttt tgaagcaact catcgattga tttacggcgc 4261 taaggatgac tctggtcaga gatacctggc ctggtctgga cacagtgccc gtgtcggagc 4321 cgcgcgagat atggcccgcg ctggagtttc aataccggag atcatgcaag ctggtggctg 4381 gaccaatgta aatattgtca tgaactatat ccgtaacctg gatagtgaaa caggggcaat 4441 ggtgcgcctg ctggaagatg gcgattaagg atccgtacgc atttaagcat aaacacgcac 4501 tatgccgttc ttctcatgta tatatatata caggcaacac gcagatatag gtgcgacgtg 4561 aacagtgagc tgtatgtgcg ggtaccgagc tcctcgagga tcagacatga taagatacat 4621 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat 4681 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttatcctcga 4741 ggagctcatg agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg ccgggggact 4801 gttgggcgcc atctccttgc atgcaccatt ccttgcggcg gcggtgctca acggcctcaa 4861 cctactactg ggctgcttcc taatgcagga gtcgcataag ggagagcgtc gacctcgagg 4921 gggggcccgg tacccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa 4981 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 5041 cgagccggga gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 5101 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 5161 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 5221 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 5281 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 5341 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 5401 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 5461 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 5521 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 5581 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 5641 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 5701 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 5761 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 5821 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 5881 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 5941 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 6001 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 6061 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 6121 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 6181 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 6241 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 6301 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 6361 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 6421 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 6481 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 6541 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 6601 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 6661 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 6721 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 6781 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 6841 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 6901 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 6961 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 7021 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 7081 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa 7141 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt 7201 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag 7261 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg 7321 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat 7381 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc 7441 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga 7501 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac 7561 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact 7621 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat 7681 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa 7741 cgacggccag tgagcgcgcg taatacgact cactataggg cgaattggag ctccaccgcg 7801 gtctttcgga cttttgaaag tgatggtggt gggggaagga ttcgaacctt cgaagtcgat 7861 gacggcagat ttagagtctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga 7921 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta 7981 gg //