LOCUS Exported 7839 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION For integration of Cre recombinase at the yeast URA3 locus orthe CYC1 terminator. ACCESSION . VERSION . KEYWORDS pSS146 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7839) AUTHORS Terweij M, van Welsem T, van Deventer S, Verzijlbergen KF, Menendez-Benito V, Ontoso D, San-Segundo P, Neefjes J, van Leeuwen F TITLE Recombination-induced tag exchange (RITE) cassette series to monitor protein dynamics in Saccharomyces cerevisiae. JOURNAL G3 (Bethesda). 2013 Aug 7;3(8):1261-72. doi: 10.1534/g3.113.006213. PUBMED 23708297 REFERENCE 2 (bases 1 to 7839) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7839 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(29..51) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 151..170 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 201..416 /gene="S. cerevisiae URA3" /label=URA3 promoter CDS 417..1220 /codon_start=1 /gene="S. cerevisiae URA3" /product="orotidine-5'-phosphate decarboxylase, required for uracil biosynthesis" /label=URA3 /note="yeast auxotrophic marker, counterselectable with 5-fluoroorotic acid (5-FOA)" /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN" rep_origin complement(1351..1806) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1488..1509) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 1700..1719 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1951..1967 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" promoter 1974..1992 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind 2025..2041 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 2025..2041 /label=pBluescriptSK /note="For pBluescript vector" promoter 2133..2163 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2171..2187 /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 2195..2211 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 2230..2250 /label=T3 /note="T3 promoter, forward primer" promoter 2232..2250 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind 2857..2880 /label=GPDpro-F /note="S. cerevisiae GPD promoter, forward primer" CDS 2970..3998 /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="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEAAHRLIYGAEDDSGQ RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVVLLE DGD" terminator 5123..5370 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(5139..5157) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" promoter complement(5389..5407) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(5417..5433) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5533..5549) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5534..5550) /label=pBluescriptKS /note="For pBluescript vector" promoter complement(5579..5597) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(5618..5634) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 5642..5658 /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(5666..5696) /label=lac promoter /note="promoter for the E. coli lac operon" primer_bind complement(5849..5866) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6020..6608) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6100..6119) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6779..7639) /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 7402..7421 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7640..7744) /gene="bla" /label=AmpR promoter primer_bind 7812..7830 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc 241 ggtttccttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg 301 agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc 361 cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt 421 cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat 481 ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca 541 aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg 601 tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg 661 ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca 721 aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac 781 acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa 841 aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg 901 gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct 961 ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac 1021 ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg 1081 atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa 1141 gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa 1201 gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac 1261 aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac 1321 agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat 1381 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa 1441 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca 1501 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg 1561 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta 1621 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg 1681 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa 1741 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg 1801 gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg 1861 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg 1921 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttgtaatacg 1981 actcactata gggcgaattg gagctccacc gcggtggcgg ccgctctaga actagtggat 2041 cccccctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat 2101 gtgagttacc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcctatg 2161 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac 2221 gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tgggggcccg gaaacagcta 2281 tgaccatgat tacgccaagc ttggctgcag gtcgagttta tcattatcaa tactcgccat 2341 ttcaaagaat acgtaaataa ttaatagtag tgattttcct aactttattt agtcaaaaaa 2401 ttagcctttt aattctgctg taacccgtac atgccaaaat agggggcggg ttacacagaa 2461 tatataacac tgatggtgct tgggtgaaca ggtttattcc tggcatccac taaatataat 2521 ggagcccgct ttttaagctg gcatccagaa aaaaaaagaa tcccagcacc aaaatattgt 2581 tttcttcacc aaccatcagt tcataggtcc attctcttag cgcaactaca gagaacaggg 2641 cacaaacagg caaaaaacgg gcacaacctc aatggagtga tgcaacctgc ctggagtaaa 2701 tgatgacaca aggcaattga cccacgcatg tatctatctc attttcttac accttctatt 2761 accttctgct ctctctgatt tggaaaaagc tgaaaaaaaa ggtttaaacc agttccctga 2821 aattattccc ctacttgact aataagtata taaagacggt aggtattgat tgtaattctg 2881 taaatctatt tcttaaactt cttaaattct acttttatag ttagtctttt ttttagtttt 2941 aaaacaccaa gaacttagtt tcgtccggaa tgtccaattt actgaccgta caccaaaatt 3001 tgcctgcatt accggtcgat gcaacgagtg atgaggttcg caagaacctg atggacatgt 3061 tcagggatcg ccaggcgttt tctgagcata cctggaaaat gcttctgtcc gtttgccggt 3121 cgtgggcggc atggtgcaag ttgaataacc ggaaatggtt tcccgcagaa cctgaagatg 3181 ttcgcgatta tcttctatat cttcaggcgc gcggtctggc agtaaaaact atccagcaac 3241 atttgggcca gctaaacatg cttcatcgtc ggtccgggct gccacgacca agtgacagca 3301 atgctgtttc actggttatg cggcggatcc gaaaagaaaa cgttgatgcc ggtgaacgtg 3361 caaaacaggc tctagcgttc gaacgcactg atttcgacca ggttcgttca ctcatggaaa 3421 atagcgatcg ctgccaggat atacgtaatc tggcatttct ggggattgct tataacaccc 3481 tgttacgtat agccgaaatt gccaggatca gggttaaaga tatctcacgt actgacggtg 3541 ggagaatgtt aatccatatt ggcagaacga aaacgctggt tagcaccgca ggtgtagaga 3601 aggcacttag cctgggggta actaaactgg tcgagcgatg gatttccgtc tctggtgtag 3661 ctgatgatcc gaataactac ctgttttgcc gggtcagaaa aaatggtgtt gccgcgccat 3721 ctgccaccag ccagctatca actcgcgccc tggaagggat ttttgaagca gctcatcgac 3781 tgatttacgg cgctgaggat gactctggtc agaggtacct ggcctggtct ggacacagtg 3841 cccgtgtcgg agccgcgcga gatatggccc gcgctggagt ttcaataccg gagatcatgc 3901 aagctggtgg ctggaccaat gtaaatattg tcatgaacta tatccgtaac ctggatagtg 3961 aaacaggggc aatggtggtc ctgctggaag atggcgatct cgagccatcc gctggagaca 4021 tgagagctgc caacctttgg ccaagcccgc tcatgatcaa acgctctaag gagaacagcc 4081 tggccttgtc cctgacggcc gaccagatgg tcagtgcctt gttggatgct gagcccccca 4141 tactctattc cgagtatgat cctaccagac ccttcagtga agcttcgatg atgggcttac 4201 tgaccaacct ggcagacagg gagctggttc acatgatcaa ctgggcgaag agggtgccag 4261 gctttgtgga tttgaccctc catgatcagg tccaccttct agaatgtgcc tggctagcga 4321 tcctgatgat tggtctcgtc tggcgctcca tggaacaccc ggggaagctc ctgtttgctc 4381 ctaacttgct cctggacagg aatcaaggta aatgtgtgga aggcatggtg gagatcttcg 4441 acatgctgct ggttacatca tctcggttcc gcatgatgaa tctgcaggga gaggagtttg 4501 tgtgcctcaa atctattatt ttgcttaatt ctggagtata cacatttctg tccagcaccc 4561 tgaagtctct cgaggagaag gaccatatcc accgagtcct ggacaagatc acagacactt 4621 tgatccacct gatggccaag gcaggcctga ccctgcagca gcagcaccag cggctagccc 4681 agctcctcct cctcctctcc cacatcaggc acatgagtaa cgaaggcatg gagcatctgt 4741 acagcatgaa gtgcaagaac gtggtacccc tctatgacct gctgctggag atgctggacg 4801 cccaccgcct acatgcgccc actagtcgtg gaggggcatc cgtggaggag acggaccaaa 4861 gccacttggc cactgcgggc tctacttcat cgcattcctt gcaaaagtat tacatcacgg 4921 gggaggcaga gggtttccct gccacagtct gagtagaatg cctgatattg actcaatatc 4981 cgttgcgttt cctgtcaaaa gtatgcgtag tgctgaacat ttcgtgatga atgccaccga 5041 ggaagaagca cggcgcggtt ttgctaaagt gatgtctgag tttggcgaac tcttgggtaa 5101 ggttggaatt gtcgacctcg agtcatgtaa ttagttatgt cacgcttaca ttcacgccct 5161 ccccccacat ccgctctaac cgaaaaggaa ggagttagac aacctgaagt ctaggtccct 5221 atttattttt ttatagttat gttagtatta agaacgttat ttatatttca aatttttctt 5281 ttttttctgt acagacgcgt gtacgcatgt aacattatac tgaaaacctt gcttgagaag 5341 gttttgggac gctcgaaggc tttaatttgc ggccggtacc caattcgccc tatagtgagt 5401 cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 5461 ttacccaact taatcgcctt gcagcacatc cccctttcgc caggggctgc aggaattcga 5521 tatcaagctt atcgataccg tcgacctcga gggggggccc ggtacccagc ttttgttccc 5581 tttagtgagg gttaattccg agcttggcgt aatcatggtc atagctgttt cctgtgtgaa 5641 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct 5701 ggggtgccta atgagtgagg taactcacat taattgcgtt gcgctcactg cccgctttcc 5761 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 5821 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 5881 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 5941 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 6001 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 6061 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 6121 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 6181 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 6241 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 6301 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 6361 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 6421 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg 6481 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 6541 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 6601 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 6661 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 6721 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 6781 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 6841 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 6901 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 6961 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 7021 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 7081 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 7141 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 7201 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 7261 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 7321 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 7381 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 7441 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 7501 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 7561 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 7621 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 7681 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 7741 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat 7801 taacctataa aaataggcgt atcacgaggc cctttcgtc //