LOCUS       sequence-255873-        7683 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 7683)
  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 7683)
  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 Gcs1 R54K AxxA
FEATURES             Location/Qualifiers
     source          1..7683
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..24,25..31)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    39..55
                     /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     44..66
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     63..79
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     63..79
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     98..118
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        100..118
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        169..518
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /label=MET17 promoter
                     /note="expression is repressed by methionine"
     primer_bind     519..535
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     519..535
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1635..1651)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1636..1652)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      1654..1901
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1670..1688)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(1919..1938)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1920..1938)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1948..1965)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1948..1964)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1957..1979)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2105..2560
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2192..2211)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2402..2423
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2861..3268
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             3269..4363
                     /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(4857..4876)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4976..4998
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5036..5054)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    5096..5599
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5636..5740
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5741..5809,5810..6601)
                     /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(5959..5978)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6772..7360
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7261..7280
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7514..7531
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7648..7669
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
       61 cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc actaaaggga
      121 acaaaagctg gagctccgga tgcaagggtt cgaatccctt agctctcatt attttttgct
      181 ttttctcttg aggtcacatg atcgcaaaat ggcaaatggc acgtgaagct gtcgatattg
      241 gggaactgtg gtggttggca aatgactaat taagttagtc aaggcgccat cctcatgaaa
      301 actgtgtaac ataataaccg aagtgtcgaa aaggtggcac cttgtccaat tgaacacgct
      361 cgatgaaaaa aataagatat atataaggtt aagtaaagcg tctgttagaa aggaagtttt
      421 tcctttttct tgctctcttg tcttttcatc tactatttcc ttcgtgtaat acagggtcgt
      481 cagatacata gatacaattc tattaccccc atccatactc tagaactagt ggatccgcga
      541 tgtcagattg gaaagtggac ccagataccc gcaggcgtct tttgcaattg caaaagattg
      601 gtgcaaataa gaaatgtatg gattgtggtg cgccaaatcc acaatgggcc acgcctaagt
      661 ttggagcttt catttgcctt gaatgtgccg gtatccataa ggggcttggt gtgcatatat
      721 cttttgtaag atctatcact atggatcagt ttaaaccgga ggaacttctt cgtatggaaa
      781 aaggtggtaa cgaacctttg actgaatggt tcaagtcgca taatattgac ctgagtttac
      841 cacaaaaagt gaaatacgat aaccccgtcg cagaggatta taaagaaaag ttaacatgtc
      901 tttgcgaaga tagagtattt gaagagcgtg agcatttgga ttttgatgct tccaagttat
      961 cagcaacctc tcaaaccgct gcatccgcta cgcctggtgt tgctcaaagt cgagaaggga
     1021 caccactgga gaaccgtcga tctgcaacac cagcaaactc tagtaatggt gccaattttc
     1081 aaaaggagaa gaacgaagct tattttgctg agctgggcaa gaagaaccaa tctagaccag
     1141 atcatttgcc cccttctcaa ggtggtaaat atcagggctt tggaagtaca cccgcaaaac
     1201 ctccacaaga acggtctgca gggtccagca atactttgag cctggaaaat tttcaagctg
     1261 atcctttggg aacattgagc agaggatggg gcctcttctc cagcgctgta accaaatcgt
     1321 tcgaggacgt aaacgaaacg gttattaagc ctcacgtcca gcaatggcaa tccggagagt
     1381 tgtcagaaga aacgaagaga gcagctgccc agtttggcca aaagtttcaa gaaacgagta
     1441 gttatggatt ccaagcattt agtaacttca ctaaaaattt caatggtaat gcagaggata
     1501 gctctacagc agggaataca acccatacag aatatcaaaa gattgataat aacgataaga
     1561 agaatgaaca ggacgaggac aaagcggacg atgcctaatc cccccgggct gcaggaattc
     1621 gatatcaagc ttatcgatac cgtcgacctc gagtcatgta attagttatg tcacgcttac
     1681 attcacgccc tccccccaca tccgctctaa ccgaaaagga aggagttaga caacctgaag
     1741 tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc
     1801 aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct
     1861 tgcttgagaa ggttttggga cgctcgaagg ctttaatttg cggccggtac ccaattcgcc
     1921 ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga
     1981 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     2041 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     2101 atggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2161 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2221 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2281 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     2341 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     2401 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     2461 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     2521 tttaacgcga attttaacaa aatattaacg cttacaattt cctgatgcgg tattttctcc
     2581 ttacgcatct gtgcggtatt tcacaccgca tatcgaccgg tcgaggagaa cttctagtat
     2641 atctacatac ctaatattat tgccttatta aaaatggaat cccaacaatt acatcaaaat
     2701 ccacattctc ttcaaaatca attgtcctgt acttccttgt tcatgtgtgt tcaaaaacgt
     2761 tatatttata ggataattat actctatttc tcaacaagta attggttgtt tggccgagcg
     2821 gtctaaggcg cctgattcaa gaaatatctt gaccgcagtt aactgtggga atactcaggt
     2881 atcgtaagat gcaagagttc gaatctctta gcaaccatta tttttttcct caacataacg
     2941 agaacacaca ggggcgctat cgcacagaat caaattcgat gactggaaat tttttgttaa
     3001 tttcagaggt cgcctgacgc atataccttt ttcaactgaa aaattgggag aaaaaggaaa
     3061 ggtgagagcg ccggaaccgg cttttcatat agaatagaga agcgttcatg actaaatgct
     3121 tgcatcacaa tacttgaagt tgacaatatt atttaaggac ctattgtttt ttccaatagg
     3181 tggttagcaa tcgtcttact ttctaacttt tcttaccttt tacatttcag caatatatat
     3241 atatatattt caaggatata ccattctaat gtctgcccct aagaagatcg tcgttttgcc
     3301 aggtgaccac gttggtcaag aaatcacagc cgaagccatt aaggttctta aagctatttc
     3361 tgatgttcgt tccaatgtca agttcgattt cgaaaatcat ttaattggtg gtgctgctat
     3421 cgatgctaca ggtgttccac ttccagatga ggcgctggaa gcctccaaga aggctgatgc
     3481 cgttttgtta ggtgctgtgg gtggtcctaa atggggtacc ggtagtgtta gacctgaaca
     3541 aggtttacta aaaatccgta aagaacttca attgtacgcc aacttaagac catgtaactt
     3601 tgcatccgac tctcttttag acttatctcc aatcaagcca caatttgcta aaggtactga
     3661 cttcgttgtt gtcagagaat tagtgggagg tatttacttt ggtaagagaa aggaagacga
     3721 tggtgatggt gtcgcttggg atagtgaaca atacaccgtt ccagaagtgc aaagaatcac
     3781 aagaatggcc gctttcatgg ccctacaaca tgagccacca ttgcctattt ggtccttgga
     3841 taaagctaat gttttggcct cttcaagatt atggagaaaa actgtggagg aaaccatcaa
     3901 gaacgaattc cctacattga aggttcaaca tcaattgatt gattctgccg ccatgatcct
     3961 agttaagaac ccaacccacc taaatggtat tataatcacc agcaacatgt ttggtgatat
     4021 catctccgat gaagcctccg ttatcccagg ttccttgggt ttgttgccat ctgcgtcctt
     4081 ggcctctttg ccagacaaga acaccgcatt tggtttgtac gaaccatgcc acggttctgc
     4141 tccagatttg ccaaagaata aggtcaaccc tatcgccact atcttgtctg ctgcaatgat
     4201 gttgaaattg tcattgaact tgcctgaaga aggtaaggcc attgaagatg cagttaaaaa
     4261 ggttttggat gcaggtatca gaactggtga tttaggtggt tccaacagta ccaccgaagt
     4321 cggtgatgct gtcgccgaag aagttaagaa aatccttgct taaaaagatt ctcttttttt
     4381 atgatatttg tacataaact ttataaatga aattcataat agaaacgaca cgaaattaca
     4441 aaatggaata tgttcatagg gtagacgaaa ctatatacgc aatctacata catttatcaa
     4501 gaaggagaaa aaggaggatg taaaggaata caggtaagca aattgatact aatggctcaa
     4561 cgtgataagg aaaaagaatt gcactttaac attaatattg acaaggagga gggcaccaca
     4621 caaaaagtta ggtgtaacag aaaatcatga aactatgatt cctaatttat atattggagg
     4681 attttctcta aaaaaaaaaa aatacaacaa ataaaaaaca ctcaatgacc tgaccatttg
     4741 atggagttta agtcaatacc ttcttgaacc atttcccata atggtgaaag ttccctcaag
     4801 aattttactc tgtcagaaac ggccttaacg acgtagtcga tatggtgcac tctcagtaca
     4861 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     4921 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     4981 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     5041 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta ggacggatcg
     5101 cttgcctgta acttacacgc gcctcgtatc ttttaatgat ggaataattt gggaatttac
     5161 tctgtgttta tttattttta tgttttgtat ttggatttta gaaagtaaat aaagaaggta
     5221 gaagagttac ggaatgaaga aaaaaaaata aacaaaggtt taaaaaattt caacaaaaag
     5281 cgtactttac atatatattt attagacaag aaaagcagat taaatagata tacattcgat
     5341 taacgataag taaaatgtaa aatcacagga ttttcgtgtg tggtcttcta cacagacaag
     5401 atgaaacaat tcggcattaa tacctgagag caggaagagc aagataaaag gtagtatttg
     5461 ttggcgatcc ccctagagtc ttttacatct tcggaaaaca aaaactattt tttctttaat
     5521 ttcttttttt actttctatt tttaatttat atatttatat taaaaaattt aaattataat
     5581 tatttttata gcacgtgatg aaaaggaccc aggtggcact tttcggggaa atgtgcgcgg
     5641 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     5701 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     5761 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     5821 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     5881 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     5941 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     6001 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     6061 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     6121 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     6181 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     6241 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     6301 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     6361 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     6421 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     6481 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     6541 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     6601 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     6661 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     6721 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     6781 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     6841 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6901 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6961 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     7021 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     7081 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     7141 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     7201 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     7261 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     7321 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     7381 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     7441 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     7501 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     7561 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     7621 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
     7681 ggc
//
