LOCUS Exported 5124 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pDRAV-3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5124) 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 5124) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5124 /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 656..689 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind 750..783 /label=attB /bound_moiety="phage phi-C31 integrase" /note="minimal attB site for the phi-C31 integrase (Groth et al., 2000)" promoter 811..1310 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(846..869) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" primer_bind 1243..1262 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 1329..2354 /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(2838..2871) /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 2878..2911 /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(2936..2954) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(2975..2991) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2975..2991) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(2988..3010) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 2999..3015 /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(3023..3053) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3068..3089 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3206..3223) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3377..3965) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3457..3476) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4136..4996) /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 4759..4778 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4997..5101) /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 ttcgtatagc atacattata cgaagttatg gcgcctcagc ggccgcacaa tgcatgtagt 721 taacttctta attaactggg atcctgcggg tgccagggcg tgcccttggg ctccccgggc 781 gcgtactcca cctcacctcg agattctacc gggtagggga ggcgcttttc ccaaggcagt 841 ctggagcatg cgctttagca gccccgctgg gcacttggcg ctacacaagt ggcctctggc 901 ctcgcacaca ttccacatcc accggtaggc gccaaccggc tccgttcttt ggtggcccct 961 tcgcgccacc ttctactcct cccctagtca ggaagttccc ccccgccccg cagctcgcgt 1021 cgtgcaggac gtgacaaatg gaagtagcac gtctcactag tctcgtgcag atggacagca 1081 ccgctgagca atggaagcgg gtaggccttt ggggcagcgg ccaatagcag ctttgctcct 1141 tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc 1201 tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa 1261 agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc gggcctttcg accgatccag 1321 ccgccaccat gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa 1381 agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca 1441 gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct 1501 acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc 1561 ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg 1621 tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg 1681 ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac 1741 cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc 1801 atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc 1861 tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg 1921 atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga 1981 gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt 2041 ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag 2101 gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct 2161 tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc 2221 gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga 2281 ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga 2341 gggcaaagga atagtcgaga aattgatgat ctattaaaca ataaagatgt ccactaaaat 2401 ggaagttttt cctgtcatac tttgttaaga agggtgagaa cagagtacct acattttgaa 2461 tggaaggatt ggagctacgg gggtgggggt ggggtgggat tagataaatg cctgctcttt 2521 actgaaggct ctttactatt gctttatgat aatgtttcat agttggatat cataatttaa 2581 acaagcaaaa ccaaattaag ggccagctca ttcctcccac tcatgatcta tagatctctc 2641 gtgggatcat tgtttttctc ttgattccca ctttgtggtt ctaagtactg tggtttccaa 2701 atgtgtcagt ttcatagcct gaagaacgag atcagcagcc tctgttccac atacacttca 2761 ttctcagtat tgttacgcgt gtgccccaac tggggtaacc tttgagttct ctcagttggg 2821 ggcgtagggt cgtcgacata acttcgtata aagtatccta tacgaagtta tatcgatgaa 2881 gttcctattc tctagaaagt ataggaactt caagcttggt acccagcttt tgttcccttt 2941 agtgagggtt aatttcgagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt 3001 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg 3061 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt 3121 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt 3181 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 3241 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 3301 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 3361 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 3421 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 3481 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 3541 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 3601 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 3661 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 3721 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 3781 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc 3841 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 3901 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 3961 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 4021 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt 4081 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc 4141 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 4201 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg 4261 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc 4321 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta 4381 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg 4441 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 4501 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 4561 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg 4621 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga 4681 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt 4741 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca 4801 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt 4861 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 4921 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 4981 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt 5041 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 5101 gcacatttcc ccgaaaagtg ccac //