LOCUS Exported 5129 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pDRAV-2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5129) 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 5129) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5129 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin complement(3..458) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(140..161) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 352..371 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 585..607 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 599..616 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 600..616 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 623..642 /label=T7 /note="T7 promoter, forward primer" promoter 623..641 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind complement(656..689) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind 756..789 /label=attB /bound_moiety="phage phi-C31 integrase" /note="minimal attB site for the phi-C31 integrase (Groth et al., 2000)" promoter 817..1316 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(852..875) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" primer_bind 1249..1268 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 1335..2360 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" protein_bind complement(2844..2877) /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." protein_bind complement(2884..2917) /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" promoter complement(2941..2959) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(2980..2996) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2980..2996) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(2993..3015) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3004..3020 /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(3028..3058) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3073..3094 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3211..3228) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3382..3970) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3462..3481) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4141..5001) /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 4764..4783 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5002..5106) /gene="bla" /label=AmpR promoter ORIGIN 1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg agctcataac 661 ttcgtataat gtatgctata cgaagttatg acatcggcgc ctcagcggcc gcacaatgca 721 tgtagttaac ttcttaatta actgggatcc tgcgggtgcc agggcgtgcc cttgggctcc 781 ccgggcgcgt actccacctc acctcgagat tctaccgggt aggggaggcg cttttcccaa 841 ggcagtctgg agcatgcgct ttagcagccc cgctgggcac ttggcgctac acaagtggcc 901 tctggcctcg cacacattcc acatccaccg gtaggcgcca accggctccg ttctttggtg 961 gccccttcgc gccaccttct actcctcccc tagtcaggaa gttccccccc gccccgcagc 1021 tcgcgtcgtg caggacgtga caaatggaag tagcacgtct cactagtctc gtgcagatgg 1081 acagcaccgc tgagcaatgg aagcgggtag gcctttgggg cagcggccaa tagcagcttt 1141 gctccttcgc tttctgggct cagaggctgg gaaggggtgg gtccgggggc gggctcaggg 1201 gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca ttctgcacgc 1261 ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc atctccgggc ctttcgaccg 1321 atccagccgc caccatgaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga 1381 tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg 1441 ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg 1501 gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg 1561 aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac 1621 agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg 1681 cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat 1741 tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg 1801 atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc 1861 aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc 1921 acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg 1981 actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga 2041 ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc 2101 ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc 2161 agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa 2221 tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg 2281 tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc 2341 gtccgagggc aaaggaatag tcgagaaatt gatgatctat taaacaataa agatgtccac 2401 taaaatggaa gtttttcctg tcatactttg ttaagaaggg tgagaacaga gtacctacat 2461 tttgaatgga aggattggag ctacgggggt gggggtgggg tgggattaga taaatgcctg 2521 ctctttactg aaggctcttt actattgctt tatgataatg tttcatagtt ggatatcata 2581 atttaaacaa gcaaaaccaa attaagggcc agctcattcc tcccactcat gatctataga 2641 tctctcgtgg gatcattgtt tttctcttga ttcccacttt gtggttctaa gtactgtggt 2701 ttccaaatgt gtcagtttca tagcctgaag aacgagatca gcagcctctg ttccacatac 2761 acttcattct cagtattgtt acgcgtgtgc cccaactggg gtaacctttg agttctctca 2821 gttgggggcg tagggtcgtc gacataactt cgtataaagt atcctatacg aagttatatc 2881 gatgaagttc ctatactttc tagagaatag gaacttcaag cttggtacca gcttttgttc 2941 cctttagtga gggttaattt cgagcttggc gtaatcatgg tcatagctgt ttcctgtgtg 3001 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc 3061 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt 3121 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg 3181 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 3241 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc 3301 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 3361 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 3421 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 3481 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 3541 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag 3601 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 3661 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 3721 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 3781 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg 3841 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 3901 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 3961 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 4021 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 4081 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 4141 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 4201 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 4261 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 4321 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 4381 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 4441 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 4501 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 4561 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 4621 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 4681 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 4741 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct 4801 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 4861 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 4921 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 4981 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 5041 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 5101 tccgcgcaca tttccccgaa aagtgccac //