LOCUS Exported 7915 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION For integration of Cre recombinase at the yeast HIS3 locus orthe CYC1 terminator. ACCESSION . VERSION . KEYWORDS pTW040 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7915) 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 7915) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7915 /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 317..504 /gene="S. cerevisiae HIS3" /label=HIS3 promoter CDS 505..1167 /codon_start=1 /gene="S. cerevisiae HIS3" /product="imidazoleglycerol-phosphate dehydratase, required for histidine biosynthesis" /label=HIS3 /note="yeast auxotrophic marker" /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM" rep_origin complement(1427..1882) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1564..1585) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 1776..1795 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2027..2043 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" promoter 2050..2068 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind 2101..2117 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 2101..2117 /label=pBluescriptSK /note="For pBluescript vector" promoter 2209..2239 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2247..2263 /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 2271..2287 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 2306..2326 /label=T3 /note="T3 promoter, forward primer" promoter 2308..2326 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind 2933..2956 /label=GPDpro-F /note="S. cerevisiae GPD promoter, forward primer" CDS 3046..4074 /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 5199..5446 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(5215..5233) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" promoter complement(5465..5483) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(5493..5509) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5609..5625) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5610..5626) /label=pBluescriptKS /note="For pBluescript vector" promoter complement(5655..5673) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(5694..5710) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 5718..5734 /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(5742..5772) /label=lac promoter /note="promoter for the E. coli lac operon" primer_bind complement(5925..5942) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6096..6684) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6176..6195) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6855..7715) /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 7478..7497 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7716..7820) /gene="bla" /label=AmpR promoter primer_bind 7888..7906 /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 accataaatt cccgttttaa gagcttggtg agcgctagga gtcactgcca ggtatcgttt 241 gaacacggca ttagtcaggg aagtcataac acagtccttt cccgcaattt tctttttcta 301 ttactcttgg cctcctctag tacactctat atttttttat gcctcggtaa tgattttcat 361 tttttttttt ccacctagcg gatgactctt tttttttctt agcgattggc attatcacat 421 aatgaattat acattatata aagtaatgtg atttcttcga agaatatact aaaaaatgag 481 caggcaagat aaacgaaggc aaagatgaca gagcagaaag ccctagtaaa gcgtattaca 541 aatgaaacca agattcagat tgcgatctct ttaaagggtg gtcccctagc gatagagcac 601 tcgatcttcc cagaaaaaga ggcagaagca gtagcagaac aggccacaca atcgcaagtg 661 attaacgtcc acacaggtat agggtttctg gaccatatga tacatgctct ggccaagcat 721 tccggctggt cgctaatcgt tgagtgcatt ggtgacttac acatagacga ccatcacacc 781 actgaagact gcgggattgc tctcggtcaa gcttttaaag aggccctagg ggccgtgcgt 841 ggagtaaaaa ggtttggatc aggatttgcg cctttggatg aggcactttc cagagcggtg 901 gtagatcttt cgaacaggcc gtacgcagtt gtcgaacttg gtttgcaaag ggagaaagta 961 ggagatctct cttgcgagat gatcccgcat tttcttgaaa gctttgcaga ggctagcaga 1021 attaccctcc acgttgattg tctgcgaggc aagaatgatc atcaccgtag tgagagtgcg 1081 ttcaaggctc ttgcggttgc cataagagaa gccacctcgc ccaatggtac caacgatgtt 1141 ccctccacca aaggtgttct tatgtagtga caccgattat ttaaagctgc agcatacgat 1201 atatatacat gtgtatatat gtatacctat gaatgtcagt aagtatgtat acgaacagta 1261 tgatactgaa gatgacaagg taatgcatca ttctatacgt gtcattctga acgaggcgcg 1321 ctttcctttt ttctttttgc tttttctttt tttttctctt gaactcgacg gatctatgcg 1381 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaaacgtt 1441 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag 1501 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt 1561 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga 1621 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg 1681 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct 1741 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc 1801 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt 1861 aatgcgccgc tacagggcgc gtcgcgccat tcgccattca ggctgcgcaa ctgttgggaa 1921 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca 1981 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc 2041 agtgaattgt aatacgactc actatagggc gaattggagc tccaccgcgg tggcggccgc 2101 tctagaacta gtggatcccc cctggcacga caggtttccc gactggaaag cgggcagtga 2161 gcgcaacgca attaatgtga gttacctcac tcattaggca ccccaggctt tacactttat 2221 gcttccggct cctatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag 2281 ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctggg 2341 ggcccggaaa cagctatgac catgattacg ccaagcttgg ctgcaggtcg agtttatcat 2401 tatcaatact cgccatttca aagaatacgt aaataattaa tagtagtgat tttcctaact 2461 ttatttagtc aaaaaattag ccttttaatt ctgctgtaac ccgtacatgc caaaataggg 2521 ggcgggttac acagaatata taacactgat ggtgcttggg tgaacaggtt tattcctggc 2581 atccactaaa tataatggag cccgcttttt aagctggcat ccagaaaaaa aaagaatccc 2641 agcaccaaaa tattgttttc ttcaccaacc atcagttcat aggtccattc tcttagcgca 2701 actacagaga acagggcaca aacaggcaaa aaacgggcac aacctcaatg gagtgatgca 2761 acctgcctgg agtaaatgat gacacaaggc aattgaccca cgcatgtatc tatctcattt 2821 tcttacacct tctattacct tctgctctct ctgatttgga aaaagctgaa aaaaaaggtt 2881 taaaccagtt ccctgaaatt attcccctac ttgactaata agtatataaa gacggtaggt 2941 attgattgta attctgtaaa tctatttctt aaacttctta aattctactt ttatagttag 3001 tctttttttt agttttaaaa caccaagaac ttagtttcgt ccggaatgtc caatttactg 3061 accgtacacc aaaatttgcc tgcattaccg gtcgatgcaa cgagtgatga ggttcgcaag 3121 aacctgatgg acatgttcag ggatcgccag gcgttttctg agcatacctg gaaaatgctt 3181 ctgtccgttt gccggtcgtg ggcggcatgg tgcaagttga ataaccggaa atggtttccc 3241 gcagaacctg aagatgttcg cgattatctt ctatatcttc aggcgcgcgg tctggcagta 3301 aaaactatcc agcaacattt gggccagcta aacatgcttc atcgtcggtc cgggctgcca 3361 cgaccaagtg acagcaatgc tgtttcactg gttatgcggc ggatccgaaa agaaaacgtt 3421 gatgccggtg aacgtgcaaa acaggctcta gcgttcgaac gcactgattt cgaccaggtt 3481 cgttcactca tggaaaatag cgatcgctgc caggatatac gtaatctggc atttctgggg 3541 attgcttata acaccctgtt acgtatagcc gaaattgcca ggatcagggt taaagatatc 3601 tcacgtactg acggtgggag aatgttaatc catattggca gaacgaaaac gctggttagc 3661 accgcaggtg tagagaaggc acttagcctg ggggtaacta aactggtcga gcgatggatt 3721 tccgtctctg gtgtagctga tgatccgaat aactacctgt tttgccgggt cagaaaaaat 3781 ggtgttgccg cgccatctgc caccagccag ctatcaactc gcgccctgga agggattttt 3841 gaagcagctc atcgactgat ttacggcgct gaggatgact ctggtcagag gtacctggcc 3901 tggtctggac acagtgcccg tgtcggagcc gcgcgagata tggcccgcgc tggagtttca 3961 ataccggaga tcatgcaagc tggtggctgg accaatgtaa atattgtcat gaactatatc 4021 cgtaacctgg atagtgaaac aggggcaatg gtggtcctgc tggaagatgg cgatctcgag 4081 ccatccgctg gagacatgag agctgccaac ctttggccaa gcccgctcat gatcaaacgc 4141 tctaaggaga acagcctggc cttgtccctg acggccgacc agatggtcag tgccttgttg 4201 gatgctgagc cccccatact ctattccgag tatgatccta ccagaccctt cagtgaagct 4261 tcgatgatgg gcttactgac caacctggca gacagggagc tggttcacat gatcaactgg 4321 gcgaagaggg tgccaggctt tgtggatttg accctccatg atcaggtcca ccttctagaa 4381 tgtgcctggc tagggatcct gatgattggt ctcgtctggc gctccatgga acacccgggg 4441 aagctcctgt ttgctcctaa cttgctcctg gacaggaatc aaggtaaatg tgtggaaggc 4501 atggtggaga tcttcgacat gctgctggtt acatcatctc ggttccgcat gatgaatctg 4561 cagggagagg agtttgtgtg cctcaaatct attattttgc ttaattctgg agtatacaca 4621 tttctgtcca gcaccctgaa gtctctcgag gagaaggacc atatccaccg agtcctggac 4681 aagatcacag acactttgat ccacctgatg gccaaggcag gcctgaccct gcagcagcag 4741 caccagcggc tagcccagct cctcctcctc ctctcccaca tcaggcacat gagtaacgaa 4801 ggcatggagc atctgtacag catgaagtgc aagaacgtgg tacccctcta tgacctgctg 4861 ctggagatgc tggacgccca ccgcctacat gcgcccacta gtcgtggagg ggcatccgtg 4921 gaggagacgg accaaagcca cttggccact gcgggctcta cttcatcgca ttccttgcaa 4981 aagtattaca tcacggggga ggcagagggt ttccctgcca cagtctgagt agaatgcctg 5041 atattgactc aatatccgtt gcgtttcctg tcaaaagtat gcgtagtgct gaacatttcg 5101 tgatgaatgc caccgaggaa gaagcacggc gcggttttgc taaagtgatg tctgagtttg 5161 gcgaactctt gggtaaggtt ggaattgtcg acctcgagtc atgtaattag ttatgtcacg 5221 cttacattca cgccctcccc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc 5281 tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa cgttatttat 5341 atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa 5401 aaccttgctt gagaaggttt tgggacgctc gaaggcttta atttgcggcc ggtacccaat 5461 tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac 5521 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagg 5581 ggctgcagga attcgatatc aagcttatcg ataccgtcga cctcgagggg gggcccggta 5641 cccagctttt gttcccttta gtgagggtta attccgagct tggcgtaatc atggtcatag 5701 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc 5761 ataaagtgta aagcctgggg tgcctaatga gtgaggtaac tcacattaat tgcgttgcgc 5821 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa 5881 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg 5941 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 6001 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 6061 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 6121 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 6181 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 6241 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 6301 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 6361 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 6421 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 6481 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca 6541 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 6601 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 6661 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 6721 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 6781 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 6841 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 6901 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 6961 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 7021 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 7081 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 7141 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt 7201 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 7261 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 7321 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 7381 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 7441 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga 7501 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 7561 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 7621 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 7681 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 7741 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 7801 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc 7861 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtk //