LOCUS       sequence-256069-        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 L246D
FEATURES             Location/Qualifiers
     source          1..7683
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..350
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /label=MET17 promoter
                     /note="expression is repressed by methionine"
     primer_bind     351..367
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     351..367
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1467..1483)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1468..1484)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      1486..1733
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1502..1520)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(1751..1770)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1752..1770)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1780..1797)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1780..1796)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1789..1811)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1937..2392
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2024..2043)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2234..2255
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2693..3100
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             3101..4195
                     /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(4689..4708)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4808..4830
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4868..4886)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4928..5431
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5468..5572
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5573..5641,5642..6433)
                     /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(5791..5810)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6604..7192
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7093..7112
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7346..7363
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7480..7501
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7516..7521,7522..7539,7540..7546)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7554..7570
                     /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     7559..7581
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7578..7594
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7578..7594
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7613..7633
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7615..7633
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ttattttttg ctttttctct tgaggtcaca tgatcgcaaa atggcaaatg gcacgtgaag
       61 ctgtcgatat tggggaactg tggtggttgg caaatgacta attaagttag tcaaggcgcc
      121 atcctcatga aaactgtgta acataataac cgaagtgtcg aaaaggtggc accttgtcca
      181 attgaacacg ctcgatgaaa aaaataagat atatataagg ttaagtaaag cgtctgttag
      241 aaaggaagtt tttccttttt cttgctctct tgtcttttca tctactattt ccttcgtgta
      301 atacagggtc gtcagataca tagatacaat tctattaccc ccatccatac tctagaacta
      361 gtggatccgc gatgtcagat tggaaagtgg acccagatac ccgcaggcgt cttttgcaat
      421 tgcaaaagat tggtgcaaat aagaaatgta tggattgtgg tgcgccaaat ccacaatggg
      481 ccacgcctaa gtttggagct ttcatttgcc ttgaatgtgc cggtatccat aaggggcttg
      541 gtgtgcatat atcttttgta agatctatca ctatggatca gtttaaaccg gaggaacttc
      601 ttcgtatgga aaaaggtggt aacgaacctt tgactgaatg gttcaagtcg cataatattg
      661 acctgagttt accacaaaaa gtgaaatacg ataaccccgt cgcagaggat tataaagaaa
      721 agttaacatg tctttgcgaa gatagagtat ttgaagagcg tgagcatttg gattttgatg
      781 cttccaagtt atcagcaacc tctcaaaccg ctgcatccgc tacgcctggt gttgctcaaa
      841 gtcgagaagg gacaccactg gagaaccgtc gatctgcaac accagcaaac tctagtaatg
      901 gtgccaattt tcaaaaggag aagaacgaag cttattttgc tgagctgggc aagaagaacc
      961 aatctagacc agatcatttg cccccttctc aaggtggtaa atatcagggc tttggaagta
     1021 cacccgcaaa acctccacaa gaacggtctg cagggtccag caatactttg agcctggaaa
     1081 attttcaagc tgatcctttg ggaacagaca gcagaggatg gggcctcttc tccagcgctg
     1141 taaccaaatc gttcgaggac gtaaacgaaa cggttattaa gcctcacgtc cagcaatggc
     1201 aatccggaga gttgtcagaa gaaacgaaga gagcagctgc ccagtttggc caaaagtttc
     1261 aagaaacgag tagttatgga ttccaagcat ttagtaactt cactaaaaat ttcaatggta
     1321 atgcagagga tagctctaca gcagggaata caacccatac agaatatcaa aagattgata
     1381 ataacgataa gaagaatgaa caggacgagg acaaatggga cgatttctaa tccccccggg
     1441 ctgcaggaat tcgatatcaa gcttatcgat accgtcgacc tcgagtcatg taattagtta
     1501 tgtcacgctt acattcacgc cctcccccca catccgctct aaccgaaaag gaaggagtta
     1561 gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta ttaagaacgt
     1621 tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca tgtaacatta
     1681 tactgaaaac cttgcttgag aaggttttgg gacgctcgaa ggctttaatt tgcggccggt
     1741 acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg
     1801 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     1861 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     1921 cctgaatggc gaatggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     1981 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     2041 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     2101 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     2161 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     2221 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     2281 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     2341 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttcctgatgc
     2401 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatcgacc ggtcgaggag
     2461 aacttctagt atatctacat acctaatatt attgccttat taaaaatgga atcccaacaa
     2521 ttacatcaaa atccacattc tcttcaaaat caattgtcct gtacttcctt gttcatgtgt
     2581 gttcaaaaac gttatattta taggataatt atactctatt tctcaacaag taattggttg
     2641 tttggccgag cggtctaagg cgcctgattc aagaaatatc ttgaccgcag ttaactgtgg
     2701 gaatactcag gtatcgtaag atgcaagagt tcgaatctct tagcaaccat tatttttttc
     2761 ctcaacataa cgagaacaca caggggcgct atcgcacaga atcaaattcg atgactggaa
     2821 attttttgtt aatttcagag gtcgcctgac gcatatacct ttttcaactg aaaaattggg
     2881 agaaaaagga aaggtgagag cgccggaacc ggcttttcat atagaataga gaagcgttca
     2941 tgactaaatg cttgcatcac aatacttgaa gttgacaata ttatttaagg acctattgtt
     3001 ttttccaata ggtggttagc aatcgtctta ctttctaact tttcttacct tttacatttc
     3061 agcaatatat atatatatat ttcaaggata taccattcta atgtctgccc ctaagaagat
     3121 cgtcgttttg ccaggtgacc acgttggtca agaaatcaca gccgaagcca ttaaggttct
     3181 taaagctatt tctgatgttc gttccaatgt caagttcgat ttcgaaaatc atttaattgg
     3241 tggtgctgct atcgatgcta caggtgttcc acttccagat gaggcgctgg aagcctccaa
     3301 gaaggctgat gccgttttgt taggtgctgt gggtggtcct aaatggggta ccggtagtgt
     3361 tagacctgaa caaggtttac taaaaatccg taaagaactt caattgtacg ccaacttaag
     3421 accatgtaac tttgcatccg actctctttt agacttatct ccaatcaagc cacaatttgc
     3481 taaaggtact gacttcgttg ttgtcagaga attagtggga ggtatttact ttggtaagag
     3541 aaaggaagac gatggtgatg gtgtcgcttg ggatagtgaa caatacaccg ttccagaagt
     3601 gcaaagaatc acaagaatgg ccgctttcat ggccctacaa catgagccac cattgcctat
     3661 ttggtccttg gataaagcta atgttttggc ctcttcaaga ttatggagaa aaactgtgga
     3721 ggaaaccatc aagaacgaat tccctacatt gaaggttcaa catcaattga ttgattctgc
     3781 cgccatgatc ctagttaaga acccaaccca cctaaatggt attataatca ccagcaacat
     3841 gtttggtgat atcatctccg atgaagcctc cgttatccca ggttccttgg gtttgttgcc
     3901 atctgcgtcc ttggcctctt tgccagacaa gaacaccgca tttggtttgt acgaaccatg
     3961 ccacggttct gctccagatt tgccaaagaa taaggtcaac cctatcgcca ctatcttgtc
     4021 tgctgcaatg atgttgaaat tgtcattgaa cttgcctgaa gaaggtaagg ccattgaaga
     4081 tgcagttaaa aaggttttgg atgcaggtat cagaactggt gatttaggtg gttccaacag
     4141 taccaccgaa gtcggtgatg ctgtcgccga agaagttaag aaaatccttg cttaaaaaga
     4201 ttctcttttt ttatgatatt tgtacataaa ctttataaat gaaattcata atagaaacga
     4261 cacgaaatta caaaatggaa tatgttcata gggtagacga aactatatac gcaatctaca
     4321 tacatttatc aagaaggaga aaaaggagga tgtaaaggaa tacaggtaag caaattgata
     4381 ctaatggctc aacgtgataa ggaaaaagaa ttgcacttta acattaatat tgacaaggag
     4441 gagggcacca cacaaaaagt taggtgtaac agaaaatcat gaaactatga ttcctaattt
     4501 atatattgga ggattttctc taaaaaaaaa aaaatacaac aaataaaaaa cactcaatga
     4561 cctgaccatt tgatggagtt taagtcaata ccttcttgaa ccatttccca taatggtgaa
     4621 agttccctca agaattttac tctgtcagaa acggccttaa cgacgtagtc gatatggtgc
     4681 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     4741 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     4801 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     4861 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     4921 taggacggat cgcttgcctg taacttacac gcgcctcgta tcttttaatg atggaataat
     4981 ttgggaattt actctgtgtt tatttatttt tatgttttgt atttggattt tagaaagtaa
     5041 ataaagaagg tagaagagtt acggaatgaa gaaaaaaaaa taaacaaagg tttaaaaaat
     5101 ttcaacaaaa agcgtacttt acatatatat ttattagaca agaaaagcag attaaataga
     5161 tatacattcg attaacgata agtaaaatgt aaaatcacag gattttcgtg tgtggtcttc
     5221 tacacagaca agatgaaaca attcggcatt aatacctgag agcaggaaga gcaagataaa
     5281 aggtagtatt tgttggcgat ccccctagag tcttttacat cttcggaaaa caaaaactat
     5341 tttttcttta atttcttttt ttactttcta tttttaattt atatatttat attaaaaaat
     5401 ttaaattata attattttta tagcacgtga tgaaaaggac ccaggtggca cttttcgggg
     5461 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     5521 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     5581 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     5641 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     5701 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     5761 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     5821 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     5881 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     5941 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     6001 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     6061 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     6121 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     6181 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     6241 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     6301 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     6361 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     6421 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     6481 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     6541 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     6601 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     6661 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     6721 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     6781 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     6841 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     6901 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     6961 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     7021 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     7081 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     7141 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     7201 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     7261 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     7321 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     7381 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     7441 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
     7501 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     7561 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa
     7621 ccctcactaa agggaacaaa agctggagct ccggatgcaa gggttcgaat cccttagctc
     7681 tca
//
