LOCUS       sequence-256091-        8106 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression S.cerevisiae.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8106)
  AUTHORS   Arakel EC, Huranova M, Estrada AF, Rau EM, Spang A, Schwappach B
  TITLE     Dissection of GTPase-activating proteins reveals functional 
            asymmetry in the COPI coat of budding yeast.
  JOURNAL   J Cell Sci. 2019 Aug 29;132(16). pii: jcs.232124. doi: 
            10.1242/jcs.232124.
  PUBMED    31331965
REFERENCE   2  (bases 1 to 8106)
  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: "J Cell 
            Sci."; date: "2019-08-29"; volume: "132(16). pii: jcs.232124. doi"; 
            pages: " 10.1242/jcs.232124"
COMMENT     Sequence Label: p415 Glo3 R59K S389,398 A
FEATURES             Location/Qualifiers
     source          1..8106
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     19..39
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        21..39
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        90..439
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /label=MET17 promoter
                     /note="expression is repressed by methionine"
     primer_bind     440..456
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     440..456
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1979..1995)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1980..1996)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      1998..2245
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2014..2032)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2263..2282)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2264..2282)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2292..2309)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2292..2308)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2301..2323)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2449..2904
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2536..2555)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2746..2767
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3205..3612
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             3613..4707
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(5201..5220)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5320..5342
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5380..5398)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    5440..5943
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5980..6084
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6085..6153,6154..6945)
                     /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(6303..6322)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7116..7704
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7605..7624
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7858..7875
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7992..8013
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8028..8033,8034..8051,8052..8058)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8066..8082
                     /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     8071..8093
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8090..8106
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8090..8106
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctccgg
       61 atgcaagggt tcgaatccct tagctctcat tattttttgc tttttctctt gaggtcacat
      121 gatcgcaaaa tggcaaatgg cacgtgaagc tgtcgatatt ggggaactgt ggtggttggc
      181 aaatgactaa ttaagttagt caaggcgcca tcctcatgaa aactgtgtaa cataataacc
      241 gaagtgtcga aaaggtggca ccttgtccaa ttgaacacgc tcgatgaaaa aaataagata
      301 tatataaggt taagtaaagc gtctgttaga aaggaagttt ttcctttttc ttgctctctt
      361 gtcttttcat ctactatttc cttcgtgtaa tacagggtcg tcagatacat agatacaatt
      421 ctattacccc catccatact ctagaactag tggatccgcg atgagtaacg atgaaggaga
      481 aacatttgcc acggaacaga ctactcaaca agtttttcag aagctaggta gcaacatgga
      541 aaatcgagtt tgttttgatt gtggtaacaa gaatcctact tggacttcgg taccttttgg
      601 tgtgatgctg tgtattcaat gctctgctgt gcataagaac atgggtgttc atatcacctt
      661 tgtaaagtct tctacattag ataaatggac aattaacaat ttaagaaggt ttaagttggg
      721 tgggaaccat aaggctcgtg attttttctt aaagaacaat gggaaacagc tactaaatac
      781 agcaaatgtt gacgcgaaaa caaaatatac gagccctgtt gctaagaaat ataagataca
      841 tctcgacaag aaggttcaaa aggatatgga gttataccca tcagaattag tcttgaacgg
      901 tcaagattct tcggactcac cgttggacac cgattcagac gcttccaggt cgacttccaa
      961 agaaaatagt gttgacgact ttttctctaa ttggcaaaaa ccaagctcca attccagttc
     1021 taaactcaat gtaaatacag gctctctcgc cccaaaaaac aacaccacag gcagcacacc
     1081 caagaccact gtcactaaaa cgagaagttc catcttaacg gctagcagaa agaaaccggt
     1141 attaaactcg caagataaaa agaaacattc aattttgtct tcgtcaagaa agcccactag
     1201 actaaccgca aagaaagtcg acaaatcaca ggcagaagac ttgtttgatc aatttaaaaa
     1261 agaggctcaa caagaaaagg aggacgagtt cactaattcg tcttctagca ccaaaattag
     1321 acaaaatgat tacgattccc aatttatgaa taacagtaaa ggcaacaata ataacagtat
     1381 agatgatatt aatacccagc ctgatgaatt caatgatttt ttaaatgaca cttcaaacag
     1441 cttcgacaca actagaaaag aacagcaaga tactttaaca cccaagtttg ccaaattggg
     1501 atttggtatg accatgaatg acgccaatga tttggcaaag caacaaaaag aatctcagaa
     1561 aatagctcag ggaccaagat atacgggcag aatcgcggaa aggtacggca cacaaaaagc
     1621 tatagcatct gatcaattat ttggtcgtgg cgcatttgat gaagcagcaa acagggaagc
     1681 gcatgacaag ttgaaaacat ttgacaatgc tacatcaatt tcctcttctt catactttgg
     1741 agaagataaa gaagtggatg aatttggtaa tcccatcaat agcagtggaa gtggagcagg
     1801 gaattttgat ggccgtaatt ctaataacgg tttcatcgat tttaatgcct ccgcagatga
     1861 tgaattgcag atgttgagag acgttgtaga acagggtgct gaaaaactcg gtagttattt
     1921 aagggactac ttaagaaaat aatccccccg ggctgcagga attcgatatc aagcttatcg
     1981 ataccgtcga cctcgagtca tgtaattagt tatgtcacgc ttacattcac gccctccccc
     2041 cacatccgct ctaaccgaaa aggaaggagt tagacaacct gaagtctagg tccctattta
     2101 tttttttata gttatgttag tattaagaac gttatttata tttcaaattt ttcttttttt
     2161 tctgtacaga cgcgtgtacg catgtaacat tatactgaaa accttgcttg agaaggtttt
     2221 gggacgctcg aaggctttaa tttgcggccg gtacccaatt cgccctatag tgagtcgtat
     2281 tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     2341 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     2401 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggac gcgccctgta
     2461 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2521 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2581 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     2641 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     2701 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     2761 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     2821 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     2881 acaaaatatt aacgcttaca atttcctgat gcggtatttt ctccttacgc atctgtgcgg
     2941 tatttcacac cgcatatcga ccggtcgagg agaacttcta gtatatctac atacctaata
     3001 ttattgcctt attaaaaatg gaatcccaac aattacatca aaatccacat tctcttcaaa
     3061 atcaattgtc ctgtacttcc ttgttcatgt gtgttcaaaa acgttatatt tataggataa
     3121 ttatactcta tttctcaaca agtaattggt tgtttggccg agcggtctaa ggcgcctgat
     3181 tcaagaaata tcttgaccgc agttaactgt gggaatactc aggtatcgta agatgcaaga
     3241 gttcgaatct cttagcaacc attatttttt tcctcaacat aacgagaaca cacaggggcg
     3301 ctatcgcaca gaatcaaatt cgatgactgg aaattttttg ttaatttcag aggtcgcctg
     3361 acgcatatac ctttttcaac tgaaaaattg ggagaaaaag gaaaggtgag agcgccggaa
     3421 ccggcttttc atatagaata gagaagcgtt catgactaaa tgcttgcatc acaatacttg
     3481 aagttgacaa tattatttaa ggacctattg ttttttccaa taggtggtta gcaatcgtct
     3541 tactttctaa cttttcttac cttttacatt tcagcaatat atatatatat atttcaagga
     3601 tataccattc taatgtctgc ccctaagaag atcgtcgttt tgccaggtga ccacgttggt
     3661 caagaaatca cagccgaagc cattaaggtt cttaaagcta tttctgatgt tcgttccaat
     3721 gtcaagttcg atttcgaaaa tcatttaatt ggtggtgctg ctatcgatgc tacaggtgtt
     3781 ccacttccag atgaggcgct ggaagcctcc aagaaggctg atgccgtttt gttaggtgct
     3841 gtgggtggtc ctaaatgggg taccggtagt gttagacctg aacaaggttt actaaaaatc
     3901 cgtaaagaac ttcaattgta cgccaactta agaccatgta actttgcatc cgactctctt
     3961 ttagacttat ctccaatcaa gccacaattt gctaaaggta ctgacttcgt tgttgtcaga
     4021 gaattagtgg gaggtattta ctttggtaag agaaaggaag acgatggtga tggtgtcgct
     4081 tgggatagtg aacaatacac cgttccagaa gtgcaaagaa tcacaagaat ggccgctttc
     4141 atggccctac aacatgagcc accattgcct atttggtcct tggataaagc taatgttttg
     4201 gcctcttcaa gattatggag aaaaactgtg gaggaaacca tcaagaacga attccctaca
     4261 ttgaaggttc aacatcaatt gattgattct gccgccatga tcctagttaa gaacccaacc
     4321 cacctaaatg gtattataat caccagcaac atgtttggtg atatcatctc cgatgaagcc
     4381 tccgttatcc caggttcctt gggtttgttg ccatctgcgt ccttggcctc tttgccagac
     4441 aagaacaccg catttggttt gtacgaacca tgccacggtt ctgctccaga tttgccaaag
     4501 aataaggtca accctatcgc cactatcttg tctgctgcaa tgatgttgaa attgtcattg
     4561 aacttgcctg aagaaggtaa ggccattgaa gatgcagtta aaaaggtttt ggatgcaggt
     4621 atcagaactg gtgatttagg tggttccaac agtaccaccg aagtcggtga tgctgtcgcc
     4681 gaagaagtta agaaaatcct tgcttaaaaa gattctcttt ttttatgata tttgtacata
     4741 aactttataa atgaaattca taatagaaac gacacgaaat tacaaaatgg aatatgttca
     4801 tagggtagac gaaactatat acgcaatcta catacattta tcaagaagga gaaaaaggag
     4861 gatgtaaagg aatacaggta agcaaattga tactaatggc tcaacgtgat aaggaaaaag
     4921 aattgcactt taacattaat attgacaagg aggagggcac cacacaaaaa gttaggtgta
     4981 acagaaaatc atgaaactat gattcctaat ttatatattg gaggattttc tctaaaaaaa
     5041 aaaaaataca acaaataaaa aacactcaat gacctgacca tttgatggag tttaagtcaa
     5101 taccttcttg aaccatttcc cataatggtg aaagttccct caagaatttt actctgtcag
     5161 aaacggcctt aacgacgtag tcgatatggt gcactctcag tacaatctgc tctgatgccg
     5221 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     5281 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     5341 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     5401 tataggttaa tgtcatgata ataatggttt cttaggacgg atcgcttgcc tgtaacttac
     5461 acgcgcctcg tatcttttaa tgatggaata atttgggaat ttactctgtg tttatttatt
     5521 tttatgtttt gtatttggat tttagaaagt aaataaagaa ggtagaagag ttacggaatg
     5581 aagaaaaaaa aataaacaaa ggtttaaaaa atttcaacaa aaagcgtact ttacatatat
     5641 atttattaga caagaaaagc agattaaata gatatacatt cgattaacga taagtaaaat
     5701 gtaaaatcac aggattttcg tgtgtggtct tctacacaga caagatgaaa caattcggca
     5761 ttaatacctg agagcaggaa gagcaagata aaaggtagta tttgttggcg atccccctag
     5821 agtcttttac atcttcggaa aacaaaaact attttttctt taatttcttt ttttactttc
     5881 tatttttaat ttatatattt atattaaaaa atttaaatta taattatttt tatagcacgt
     5941 gatgaaaagg acccaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     6001 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     6061 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     6121 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     6181 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     6241 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     6301 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     6361 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     6421 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     6481 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     6541 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     6601 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     6661 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     6721 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     6781 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     6841 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     6901 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     6961 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     7021 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     7081 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     7141 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     7201 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     7261 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     7321 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     7381 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     7441 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     7501 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     7561 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     7621 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     7681 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     7741 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     7801 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     7861 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     7921 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     7981 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     8041 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     8101 tatgac
//
