LOCUS       sequence-256105-        7682 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 7682)
  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 7682)
  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
FEATURES             Location/Qualifiers
     source          1..7682
                     /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(1555..1571)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1556..1572)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      1574..1821
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1590..1608)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(1839..1858)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1840..1858)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1868..1885)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1868..1884)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1877..1899)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2025..2480
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2112..2131)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2322..2343
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2781..3188
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             3189..4283
                     /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(4777..4796)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4896..4918
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4956..4974)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    5016..5519
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5556..5660
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5661..5729,5730..6521)
                     /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(5879..5898)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6692..7280
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7181..7200
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7434..7451
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7568..7589
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7604..7609,7610..7627,7628..7634)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7642..7658
                     /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     7647..7669
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7666..7682
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7666..7682
                     /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 atgtcagatt ggaaagtgga
      481 cccagatacc cgcaggcgtc ttttgcaatt gcaaaagatt ggtgcaaata agaaatgtat
      541 ggattgtggt gcgccaaatc cacaatgggc cacgcctaag tttggagctt tcatttgcct
      601 tgaatgtgcc ggtatccata gagggcttgg tgtgcatata tcttttgtaa gatctatcac
      661 tatggatcag tttaaaccgg aggaacttct tcgtatggag aaaggtggta acgaaccttt
      721 gactgaatgg ttcaagtcgc ataatattga cctgagttta ccacaaaaag tgaaatacga
      781 taaccccgtc gcagaggatt ataaagaaaa gttaacatgt ctttgcgaag atagagtatt
      841 tgaagagcgt gagcatttgg attttgatgc ttccaagtta tcagcaacct ctcaaaccgc
      901 tgcatccgct acgcctggtg ttgctcaaag tcgagaaggg acaccactgg agaaccgtcg
      961 atctgcaaca ccagcaaact ctagtaatgg tgccaatttt caaaaggaga agaacgaagc
     1021 ttattttgct gagctgggca agaagaacca atctagacca gatcatttgc ccccttctca
     1081 aggtggtaaa tatcagggct ttggaagtac acccgcaaaa cctccacaag aacggtctgc
     1141 agggtccagc aatactttga gcctggaaaa ttttcaagct gatcctttgg gaacattgag
     1201 cagaggatgg ggcctcttct ccagcgctgt aaccaaatcg ttcgaggacg taaacgaaac
     1261 ggttattaag cctcacgtcc agcaatggca atccggagag ttgtcagaag aaacgaagag
     1321 agcagctgcc cagtttggcc aaaagtttca agaaacgagt agttatggat tccaagcatt
     1381 tagtaacttc actaaaaatt tcaatggtaa tgcagaggat agctctacag cagggaatac
     1441 aacccataca gaatatcaaa agattgataa taacgataag aagaatgaac aggacgagga
     1501 caaatgggac gatttctaat cccccgggct gcaggaattc gatatcaagc ttatcgatac
     1561 cgtcgacctc gagtcatgta attagttatg tcacgcttac attcacgccc tccccccaca
     1621 tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt
     1681 tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg
     1741 tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga
     1801 cgctcgaagg ctttaatttg cggccggtac ccaattcgcc ctatagtgag tcgtattacg
     1861 cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
     1921 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
     1981 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg
     2041 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     2101 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     2161 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     2221 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     2281 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     2341 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     2401 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     2461 aatattaacg cttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     2521 tcacaccgca tatcgaccgg tcgaggagaa cttctagtat atctacatac ctaatattat
     2581 tgccttatta aaaatggaat cccaacaatt acatcaaaat ccacattctc ttcaaaatca
     2641 attgtcctgt acttccttgt tcatgtgtgt tcaaaaacgt tatatttata ggataattat
     2701 actctatttc tcaacaagta attggttgtt tggccgagcg gtctaaggcg cctgattcaa
     2761 gaaatatctt gaccgcagtt aactgtggga atactcaggt atcgtaagat gcaagagttc
     2821 gaatctctta gcaaccatta tttttttcct caacataacg agaacacaca ggggcgctat
     2881 cgcacagaat caaattcgat gactggaaat tttttgttaa tttcagaggt cgcctgacgc
     2941 atataccttt ttcaactgaa aaattgggag aaaaaggaaa ggtgagagcg ccggaaccgg
     3001 cttttcatat agaatagaga agcgttcatg actaaatgct tgcatcacaa tacttgaagt
     3061 tgacaatatt atttaaggac ctattgtttt ttccaatagg tggttagcaa tcgtcttact
     3121 ttctaacttt tcttaccttt tacatttcag caatatatat atatatattt caaggatata
     3181 ccattctaat gtctgcccct aagaagatcg tcgttttgcc aggtgaccac gttggtcaag
     3241 aaatcacagc cgaagccatt aaggttctta aagctatttc tgatgttcgt tccaatgtca
     3301 agttcgattt cgaaaatcat ttaattggtg gtgctgctat cgatgctaca ggtgttccac
     3361 ttccagatga ggcgctggaa gcctccaaga aggctgatgc cgttttgtta ggtgctgtgg
     3421 gtggtcctaa atggggtacc ggtagtgtta gacctgaaca aggtttacta aaaatccgta
     3481 aagaacttca attgtacgcc aacttaagac catgtaactt tgcatccgac tctcttttag
     3541 acttatctcc aatcaagcca caatttgcta aaggtactga cttcgttgtt gtcagagaat
     3601 tagtgggagg tatttacttt ggtaagagaa aggaagacga tggtgatggt gtcgcttggg
     3661 atagtgaaca atacaccgtt ccagaagtgc aaagaatcac aagaatggcc gctttcatgg
     3721 ccctacaaca tgagccacca ttgcctattt ggtccttgga taaagctaat gttttggcct
     3781 cttcaagatt atggagaaaa actgtggagg aaaccatcaa gaacgaattc cctacattga
     3841 aggttcaaca tcaattgatt gattctgccg ccatgatcct agttaagaac ccaacccacc
     3901 taaatggtat tataatcacc agcaacatgt ttggtgatat catctccgat gaagcctccg
     3961 ttatcccagg ttccttgggt ttgttgccat ctgcgtcctt ggcctctttg ccagacaaga
     4021 acaccgcatt tggtttgtac gaaccatgcc acggttctgc tccagatttg ccaaagaata
     4081 aggtcaaccc tatcgccact atcttgtctg ctgcaatgat gttgaaattg tcattgaact
     4141 tgcctgaaga aggtaaggcc attgaagatg cagttaaaaa ggttttggat gcaggtatca
     4201 gaactggtga tttaggtggt tccaacagta ccaccgaagt cggtgatgct gtcgccgaag
     4261 aagttaagaa aatccttgct taaaaagatt ctcttttttt atgatatttg tacataaact
     4321 ttataaatga aattcataat agaaacgaca cgaaattaca aaatggaata tgttcatagg
     4381 gtagacgaaa ctatatacgc aatctacata catttatcaa gaaggagaaa aaggaggatg
     4441 taaaggaata caggtaagca aattgatact aatggctcaa cgtgataagg aaaaagaatt
     4501 gcactttaac attaatattg acaaggagga gggcaccaca caaaaagtta ggtgtaacag
     4561 aaaatcatga aactatgatt cctaatttat atattggagg attttctcta aaaaaaaaaa
     4621 aatacaacaa ataaaaaaca ctcaatgacc tgaccatttg atggagttta agtcaatacc
     4681 ttcttgaacc atttcccata atggtgaaag ttccctcaag aattttactc tgtcagaaac
     4741 ggccttaacg acgtagtcga tatggtgcac tctcagtaca atctgctctg atgccgcata
     4801 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     4861 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     4921 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     4981 ggttaatgtc atgataataa tggtttctta ggacggatcg cttgcctgta acttacacgc
     5041 gcctcgtatc ttttaatgat ggaataattt gggaatttac tctgtgttta tttattttta
     5101 tgttttgtat ttggatttta gaaagtaaat aaagaaggta gaagagttac ggaatgaaga
     5161 aaaaaaaata aacaaaggtt taaaaaattt caacaaaaag cgtactttac atatatattt
     5221 attagacaag aaaagcagat taaatagata tacattcgat taacgataag taaaatgtaa
     5281 aatcacagga ttttcgtgtg tggtcttcta cacagacaag atgaaacaat tcggcattaa
     5341 tacctgagag caggaagagc aagataaaag gtagtatttg ttggcgatcc ccctagagtc
     5401 ttttacatct tcggaaaaca aaaactattt tttctttaat ttcttttttt actttctatt
     5461 tttaatttat atatttatat taaaaaattt aaattataat tatttttata gcacgtgatg
     5521 aaaaggaccc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5581 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5641 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5701 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5761 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5821 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     5881 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     5941 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     6001 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     6061 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     6121 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     6181 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     6241 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     6301 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     6361 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     6421 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     6481 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     6541 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6601 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6661 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6721 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6781 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     6841 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     6901 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     6961 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     7021 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     7081 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     7141 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     7201 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     7261 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     7321 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     7381 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     7441 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     7501 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     7561 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     7621 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     7681 ac
//
