LOCUS       sequence-255869-        7147 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For ScMET17 promoter regulated expression of mCherry-Cub-R-GFP (CCG)
            fused bait proteins, centromeric ARS plasmid, URA3 complements the 
            uracil auxotrophy of S. cerevisiae ura mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7147)
  AUTHORS   Dunkler A, Rosler R, Kestler HA, Moreno-Andres D, Johnsson N
  TITLE     SPLIFF: A Single-Cell Method to Map Protein-Protein Interactions in 
            Time and Space.
  JOURNAL   Methods Mol Biol. 2015;1346:151-68. doi: 
            10.1007/978-1-4939-2987-0_11.
  PUBMED    26542721
REFERENCE   2  (bases 1 to 7147)
  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"; journalName: "Methods Mol
            Biol."; date: "2015"; volume: "1346:151-68. doi"; pages: " 
            10.1007/978-1-4939-2987-0_11"
COMMENT     Sequence Label: pMET17p-CCG 316
FEATURES             Location/Qualifiers
     source          1..7147
                     /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        194..409
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             410..1213
                     /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(1344..1799)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1481..1502)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1693..1712
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1925..1947
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1939..1956
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1940..1956
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1963..1982
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1963..1981
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2183..2533
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /label=MET17 promoter
                     /note="expression is repressed by methionine"
     primer_bind     2547..2563
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2547..2563
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             2620..3327
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2765..2783)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3033..3052
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3601..3627
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3640..4353
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MGKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(3670..3698)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4293..4313
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     promoter        complement(4374..4392)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4413..4429)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4413..4429)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4426..4448)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4437..4453
                     /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(4461..4467,4468..4485,4486..4491))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4506..4527
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4644..4661)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4815..5403)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4895..4914)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5574..6365,6366..6434))
                     /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     6197..6216
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6435..6539)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    6576..7079
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     7120..7138
                     /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 cttttcaatt catcattttt tttttattct tttttttgat ttcggtttcc
      241 ttgaaatttt tttgattcgg taatctccga acagaaggaa gaacgaagga aggagcacag
      301 acttagattg gtatatatac gcatatgtag tgttgaagaa acatgaaatt gcccagtatt
      361 cttaacccaa ctgcacagaa caaaaacctg caggaaacga agataaatca tgtcgaaagc
      421 tacatataag gaacgtgctg ctactcatcc tagtcctgtt gctgccaagc tatttaatat
      481 catgcacgaa aagcaaacaa acttgtgtgc ttcattggat gttcgtacca ccaaggaatt
      541 actggagtta gttgaagcat taggtcccaa aatttgttta ctaaaaacac atgtggatat
      601 cttgactgat ttttccatgg agggcacagt taagccgcta aaggcattat ccgccaagta
      661 caatttttta ctcttcgaag acagaaaatt tgctgacatt ggtaatacag tcaaattgca
      721 gtactctgcg ggtgtataca gaatagcaga atgggcagac attacgaatg cacacggtgt
      781 ggtgggccca ggtattgtta gcggtttgaa gcaggcggca gaagaagtaa caaaggaacc
      841 tagaggcctt ttgatgttag cagaattgtc atgcaagggc tccctatcta ctggagaata
      901 tactaagggt actgttgaca ttgcgaagag cgacaaagat tttgttatcg gctttattgc
      961 tcaaagagac atgggtggaa gagatgaagg ttacgattgg ttgattatga cacccggtgt
     1021 gggtttagat gacaagggag acgcattggg tcaacagtat agaaccgtgg atgatgtggt
     1081 ctctacagga tctgacatta ttattgttgg aagaggacta tttgcaaagg gaagggatgc
     1141 taaggtagag ggtgaacgtt acagaaaagc aggctgggaa gcatatttga gaagatgcgg
     1201 ccagcaaaac taaaaaactg tattataagt aaatgcatgt atactaaact cacaaattag
     1261 agcttcaatt taattatatc agttattacc ctatgcggtg tgaaataccg cacagatgcg
     1321 taaggagaaa ataccgcatc aggaaattgt aagcgttaat attttgttaa aattcgcgtt
     1381 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     1441 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     1501 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     1561 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     1621 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     1681 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     1741 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     1801 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     1861 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     1921 ttttcccagt cacgacgttg taaaacgacg gccagtgaat tgtaatacga ctcactatag
     1981 ggcgaattgg agctccaccg cggtttcgcc atttcttgaa tgtatttaaa gatatttaat
     2041 gctataatag acatttaaat ccaattcttc caacatacaa tgggagtttg gccgagtggt
     2101 ttaaggcgtc agatttaggt ggatttaacc tctaaaatct ctgatatctt cggatgcaag
     2161 ggttcgaatc ccttagctct cattattttt tgctttttct cttgaggtca catgatcgca
     2221 aaatggcaaa tggcacgtga agctgtcgat attggggaac tgtggtggtt ggcaaatgac
     2281 taattaagtt agtcaaggcg ccatcctcat gaaaactgtg taacataata accgaagtgt
     2341 cgaaaaggtg gcaccttgtc caattgaaca cgctcgatga aaaaaataag atatatataa
     2401 ggttaagtaa agcgtctgtt agaaaggaag tttttccttt ttcttgctct cttgtctttt
     2461 catctactat ttccttcgtg taatacaggg tcgtcagata catagataca attctattac
     2521 ccccatccat acatctagac ggccgctcta gaactagtgg atcccccggg ctgcaggaat
     2581 tcgatatcaa gcttatcgat accgtcgacc ggcgcgccaa tggtgagcaa gggcgaggag
     2641 gataacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg
     2701 aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag
     2761 accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc
     2821 cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac
     2881 ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc
     2941 ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg
     3001 aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc
     3061 tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag
     3121 cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag
     3181 gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc
     3241 tcccacaacg aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc
     3301 accggcggca tggacgagct gtacaagggc tcgaccggta tccctccaga tcaacaaaga
     3361 ttgatctttg ccggtaagca gctagaagac ggtagaacgc tgtctgatta caacattcag
     3421 aaggagtcca ccttacatct tgtgctaagg ctaagaggtg gtaggcacgg atccggagct
     3481 tggctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc
     3541 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgtggc
     3601 tacccatacg acgtcccaga ttacgccgct ggcgcgccaa tgggtaaagg agaagaactt
     3661 ttcactggag ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg gcacaaattt
     3721 tctgtcagtg gagagggtga aggcgatgca acatacggaa aacttaccct taaatttatt
     3781 tgcactactg gaaaactacc tgttccatgg ccaacacttg tcactacttt cacctatggt
     3841 gttcaatgct tttcaagata cccagatcat atgaaacggc atgacttttt caagagtgcc
     3901 atgcccgaag gttatgtaca ggaaagaact atatttttca aagatgacgg gaactacaag
     3961 acacgtgctg aagtcaagtt tgaaggtgat acccttgtta atagaatcga gttaaaaggt
     4021 attgatttta aagaagatgg aaacattctt ggacacaaat tggaatacaa ctataactca
     4081 cacaatgtat acatcatggc agacaaacaa aagaatggaa tcaaagccaa cttcaaaatt
     4141 agacacaaca ttgaagatgg aagcgttcaa ctagcagacc attatcaaca aaatactcca
     4201 attggcgatg gccctgtcct tttaccagac aaccattacc tgtccacaca atctgccctt
     4261 tcgaaagatc ccaacgaaaa gagagaccac atggtccttc ttgagtttgt aacagctgct
     4321 gggattacac atggcatgga tgaactatac aaataaggta ccagcttttg ttccctttag
     4381 tgagggttaa tttcgagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     4441 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     4501 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4561 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4621 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4681 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4741 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4801 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4861 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4921 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4981 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5041 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5101 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5161 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5221 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     5281 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5341 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5401 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     5461 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     5521 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     5581 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     5641 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5701 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5761 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5821 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5881 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5941 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6001 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6061 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6121 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6181 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6241 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     6301 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     6361 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     6421 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     6481 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     6541 acatttcccc gaaaagtgcc acctgggtcc ttttcatcac gtgctataaa aataattata
     6601 atttaaattt tttaatataa atatataaat taaaaataga aagtaaaaaa agaaattaaa
     6661 gaaaaaatag tttttgtttt ccgaagatgt aaaagactct agggggatcg ccaacaaata
     6721 ctacctttta tcttgctctt cctgctctca ggtattaatg ccgaattgtt tcatcttgtc
     6781 tgtgtagaag accacacacg aaaatcctgt gattttacat tttacttatc gttaatcgaa
     6841 tgtatatcta tttaatctgc ttttcttgtc taataaatat atatgtaaag tacgcttttt
     6901 gttgaaattt tttaaacctt tgtttatttt tttttcttca ttccgtaact cttctacctt
     6961 ctttatttac tttctaaaat ccaaatacaa aacataaaaa taaataaaca cagagtaaat
     7021 tcccaaatta ttccatcatt aaaagatacg aggcgcgtgt aagttacagg caagcgatcc
     7081 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     7141 tttcgtc
//
