LOCUS       sequence-109187-        7839 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For integration of Cre recombinase at the yeast URA3 locus orthe 
            CYC1 terminator.
ACCESSION   .
VERSION     .
KEYWORDS    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1534/g3.113"; 
            journalName: "G3 (Bethesda)"; date: "2013-08-7- 7"; volume: "3"; 
            issue: "8"; pages: "1261-72"
COMMENT     Sequence Label: pSS146
FEATURES             Location/Qualifiers
     source          1..7839
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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        196..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        join(2133..2138,2139..2156,2157..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"
     promoter        2232..2250
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2321..2963
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     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"
     CDS             4011..4946
                     /codon_start=1
                     /product="mutated ligand-binding domain of the human 
                     estrogen receptor (Feil et al., 1997)"
                     /label=ERT2
                     /translation="AGDMRAANLWPSPLMIKRSKENSLALSLTADQMVSALLDAEPPIL
                     YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLAI
                     LMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLVTSSRFRMMNLQGEEF
                     VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
                     AQLLLLLSHIRHMSNEGMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEET
                     DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
     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(join(5666..5672,5673..5690,5691..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(join(6779..7570,7571..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
//
