LOCUS Exported 5630 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION integration of loxP site directly upstream of gene start site - contains S. pombe nmt1 promoter. ACCESSION . VERSION . KEYWORDS pAW32 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5630) AUTHORS Watson AT1, Garcia V, Bone N, Carr AM, Armstrong J. TITLE Gene tagging and gene replacement using recombinase-mediated cassette exchange in Schizosaccharomyces pombe. JOURNAL Gene. 2008 Jan 15;407(1-2):63-74 PUBMED 18054176 REFERENCE 2 (bases 1 to 5630) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5630 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(5..24) /label=T7 /note="T7 promoter, forward primer" promoter complement(6..24) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(31..48) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(31..47) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(40..62) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 188..643 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(275..294) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 485..506 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(710..729) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 829..851 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(889..907) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 975..1079 /gene="bla" /label=AmpR promoter CDS 1080..1940 /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(1298..1317) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2111..2699 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2600..2619 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2853..2870 /label=L4440 /note="L4440 vector, forward primer" protein_bind 2987..3008 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 3023..3053 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3061..3077 /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 3066..3088 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 3085..3101 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 3085..3101 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" promoter 3119..3137 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 3119..3136 /label=SP6 /note="SP6 promoter, forward primer" protein_bind 3154..3187 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS 3721..4515 /codon_start=1 /gene="Schizosaccharomyces pombe ura4" /product="orotidine 5'-phosphate decarboxylase, required for uracil biosynthesis" /label=S. pombe ura4 /translation="MDARVFQSYSARAEGMKNPIAKELLALMEEKQSNLSVAVDLTKKS EILELVDKIGPYVCVIKTHIDVVEDFDQDMVEKLVALGKKHRFLIFEDRKFADIGNTVK LQYASGVYKIASWAHITNCHTVSGEGIIQGLKEVGLPLGRGLLLLAEMSSKGSLATGSY TEKTLEWFEKHTDFCFGFIAGRRFPNLQSDYITMSPGIGLDVKGDGLGQQYRTPEEVIV NCGSDIIIVGRGVYGAGRNPVVEAKRYREAGWKAYQQRLSQH" protein_bind 5577..5610 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." ORIGIN 1 ttcgccctat agtgagtcgt attacaattc actggccgtc gttttacaac gtcgtgactg 61 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg 121 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg 181 cgaatggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 241 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 301 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 361 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt 421 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 481 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt 541 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 601 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttcctgatg cggtattttc 661 tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct 721 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac 781 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca 841 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac 901 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt 961 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 1021 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 1081 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 1141 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 1201 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 1261 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 1321 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 1381 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 1441 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 1501 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 1561 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 1621 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 1681 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 1741 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 1801 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 1861 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 1921 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt 1981 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 2041 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 2101 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 2161 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 2221 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag 2281 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 2341 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 2401 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 2461 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 2521 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 2581 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 2641 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 2701 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 2761 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 2821 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 2881 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 2941 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 3001 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 3061 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat 3121 ttaggtgaca ctatagaata ctcaagcttg gacataactt cgtatagcat acattatacg 3181 aagttatctg cagagctaca aatcccactg gctatatgta tgcatttgtg ttaaaaaagt 3241 ttgtatagat tatttaatct actcagcatt ctttctctaa ataggaattt gttacttaat 3301 ggagaaaaaa atgtttcgat ttacctagtg tatttgtttg tatactcacg tttaatttca 3361 aacatccatt ctatcttgtg taatttttgg catggtgaaa aagataatca gccttataat 3421 ctttacaaaa gtaagaaatt ctgtaaataa gccttaatgc ccttgcttta aattaaaatg 3481 gttctttttc atgataatgt ttgcactttg tgaatatatt ttagatagtt ctgtgaggta 3541 taattaagat gttttagaga cttatacaat tttgtcttta taaattctta attgatttta 3601 ccatcccagt ttaactatgc ttcgtcggca tctctgcaca tgtcgtgttt tcttaccgta 3661 ttgtcctacc aagaacctct tttttgcttg gatcgaaatt aaaggtttaa aagcaaagtt 3721 atggatgcta gagtatttca aagctattca gctagagctg aggggatgaa aaatcccatt 3781 gccaaggaat tgttggcttt gatggaagaa aagcaaagca acttgtcagt cgcggtcgat 3841 ttgacgaaga aatccgaaat cttagaattg gtagataaaa ttggacccta tgtctgtgtt 3901 atcaagacac atattgacgt tgtcgaggat ttcgaccagg atatggtaga aaaactggtg 3961 gccttaggta aaaagcatcg ttttcttatc tttgaggatc gcaaattcgc agacattgga 4021 aataccgtca agctacaata tgcatctggt gtgtacaaaa ttgcttcttg ggctcatatc 4081 acaaattgcc atacagtgtc aggcgagggt attatacaag gcctcaaaga agttggttta 4141 cctttgggac gtggtctctt gcttttggct gaaatgtctt ccaaaggctc tttggctact 4201 ggttcctaca cagagaaaac cttagaatgg tttgagaagc ataccgattt ttgctttggc 4261 tttatagctg gtcgtcgatt tcctaacctt caaagcgact acataactat gtcccctggt 4321 atcggcttgg atgttaaagg agacgggctg ggacagcaat atcgtactcc tgaagaagtg 4381 attgtaaact gcggtagcga tatcatcatt gttggtcgtg gagtctatgg agctggtcgt 4441 aatcctgttg tcgaagccaa gagatataga gaagctggtt ggaaggcata tcagcaaaga 4501 ctttctcagc attaaaaaaa gactaatgta aaattttttt ggttggttat tgaaaaagtc 4561 gatgccttgt ttgcgtttgt tttcctaggc gttttatgtc agaaggcatt tagaattagt 4621 atacaagtac tctttggtaa aattttatgt agcgactaaa atattaacta ttatagataa 4681 acaccttggg aataaaaagt aatttgctat agtaatttat taaacatgct cctacaacat 4741 taccacaatc ttttctcttg gattgacatt gaataagaaa agagtgaatt tttttagact 4801 tgtaatgata actatgtaca aagccaatga aagatgtatg tagatgaatg taaaatacca 4861 tgtagacaaa caagataaaa cttggttata aacattggtg ttggaacaga ataaattaga 4921 tgtcaaaaag tttcgtcaat atcacaagcc tgcagagatc tgttgaaccg aaattgcaga 4981 tactgatata tgaaaataat acccacaatt ttgggaatag cgcaagcctc aatttaaaca 5041 ataggtgagg acacatgata atgacctcaa tgattgttag aagaaaagag cctcattaca 5101 aaatcgaaaa atgaatggtt gggtacaagt ttccaaaaca tggtaaagtg gactttgcgt 5161 atgagacgta aatagaaaaa aacacttgtt atatgttttc tagaattatt gttgtctctt 5221 tatggttgga tgatgcaaaa tagtaatttc ggttagttgc tgtaaaacac cacgagacaa 5281 atagatatgg atatttatta aatcaggaaa aacgtaactc tcggctactg gatggttcag 5341 tcacccaacg attactgggg agagaaaaca gggcaaaagc aaagcttaaa ggaatccgat 5401 tgtcattcgg caatgtgcag cgaaactaaa aaccggataa tggacctgtt aatcgaaaca 5461 ttgaagatat ataaaggaag aggaatcctg gcatatcatc aattgaataa gttgaattaa 5521 ttatttcaat ctcattctca ctttctgact tatagtcgct ttgttaaatc agatctataa 5581 cttcgtatag catacattat acgaagttat cgggcggcgg gggtggtgaa //