LOCUS       sequence-256193-        7917 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 7917)
  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 7917)
  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 Delta-C
FEATURES             Location/Qualifiers
     source          1..7917
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        51..400
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /label=MET17 promoter
                     /note="expression is repressed by methionine"
     primer_bind     401..417
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     401..417
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1751..1767)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1752..1768)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      1770..2017
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1786..1804)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2035..2054)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2036..2054)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2064..2081)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2064..2080)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2073..2095)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2221..2676
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2308..2327)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2518..2539
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2977..3384
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             3385..4479
                     /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(4973..4992)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5092..5114
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5152..5170)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    5212..5715
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5752..5856
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5857..5925,5926..6717)
                     /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(6075..6094)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6888..7476
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7377..7396
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7630..7647
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7764..7785
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7800..7805,7806..7823,7824..7830)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7838..7854
                     /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     7843..7865
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7862..7878
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7862..7878
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7897..7917
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7899..7917
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 gaacaaaagc tggagctccg gatgcaaggg ttcgaatccc ttagctctca ttattttttg
       61 ctttttctct tgaggtcaca tgatcgcaaa atggcaaatg gcacgtgaag ctgtcgatat
      121 tggggaactg tggtggttgg caaatgacta attaagttag tcaaggcgcc atcctcatga
      181 aaactgtgta acataataac cgaagtgtcg aaaaggtggc accttgtcca attgaacacg
      241 ctcgatgaaa aaaataagat atatataagg ttaagtaaag cgtctgttag aaaggaagtt
      301 tttccttttt cttgctctct tgtcttttca tctactattt ccttcgtgta atacagggtc
      361 gtcagataca tagatacaat tctattaccc ccatccatac tctagaacta gtggatccgc
      421 gatgagtaac gatgaaggag aaacatttgc cacggaacag actactcaac aagtttttca
      481 gaagctaggt agcaacatgg aaaatcgagt ttgttttgat tgtggtaaca agaatcctac
      541 ttggacttcg gtaccttttg gtgtgatgct gtgtattcaa tgctctgctg tgcataagaa
      601 catgggtgtt catatcacct ttgtaaagtc ttctacatta gataaatgga caattaacaa
      661 tttaagaagg tttaagttgg gtgggaacca taaggctcgt gattttttct taaagaacaa
      721 tgggaaacag ctactaaata cagcaaatgt tgacgcgaaa acaaaatata cgagccctgt
      781 tgctaagaaa tataagatac atctcgacaa gaaggttcaa aaggatatgg agttataccc
      841 atcagaatta gtcttgaacg gtcaagattc ttcggactca ccgttggaca ccgattcaga
      901 cgcttccagg tcgacttcca aagaaaatag tgttgacgac tttttctcta attggcaaaa
      961 accaagctcc aattccagtt ctaaactcaa tgtaaataca ggctctctcg ccccaaaaaa
     1021 caacaccaca ggcagcacac ccaagaccac tgtcactaaa acgagaagtt ccatcttaac
     1081 ggctagcaga aagaaaccgg tattaaactc gcaagataaa aagaaacatt caattttgtc
     1141 ttcgtcaaga aagcccacta gactaaccgc aaagaaagtc gacaaatcac aggcagaaga
     1201 cttgtttgat caatttaaaa aagaggctca acaagaaaag gaggacgagt tcactaattc
     1261 gtcttctagc accaaaatta gacaaaatga ttacgattcc caatttatga ataacagtaa
     1321 aggcaacaat aataacagta tagatgatat taatacccag cctgatgaat tcaatgattt
     1381 tttaaatgac acttcaaaca gcttcgacac aactagaaaa gaacagcaag atactttaac
     1441 acccaagttt gccaaattgg gatttggtat gaccatgaat gacgccaatg atttggcaaa
     1501 gcaacaaaaa gaatctcaga aaatagctca gggaccaaga tatacgggca gaatcgcgga
     1561 aaggtacggc acacaaaaag ctatatcatc tgatcaatta tttggtcgtg gctcatttga
     1621 tgaagcagca aacagggaag cgcatgacaa gttgaaaaca tttgacaatg ctacatcaat
     1681 ttcctcttct tcatactttg gagaagataa ataatccccc cgggctgcag gaattcgata
     1741 tcaagcttat cgataccgtc gacctcgagt catgtaatta gttatgtcac gcttacattc
     1801 acgccctccc cccacatccg ctctaaccga aaaggaagga gttagacaac ctgaagtcta
     1861 ggtccctatt tattttttta tagttatgtt agtattaaga acgttattta tatttcaaat
     1921 ttttcttttt tttctgtaca gacgcgtgta cgcatgtaac attatactga aaaccttgct
     1981 tgagaaggtt ttgggacgct cgaaggcttt aatttgcggc cggtacccaa ttcgccctat
     2041 agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     2101 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     2161 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     2221 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     2281 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     2341 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     2401 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     2461 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     2521 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     2581 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     2641 acgcgaattt taacaaaata ttaacgctta caatttcctg atgcggtatt ttctccttac
     2701 gcatctgtgc ggtatttcac accgcatatc gaccggtcga ggagaacttc tagtatatct
     2761 acatacctaa tattattgcc ttattaaaaa tggaatccca acaattacat caaaatccac
     2821 attctcttca aaatcaattg tcctgtactt ccttgttcat gtgtgttcaa aaacgttata
     2881 tttataggat aattatactc tatttctcaa caagtaattg gttgtttggc cgagcggtct
     2941 aaggcgcctg attcaagaaa tatcttgacc gcagttaact gtgggaatac tcaggtatcg
     3001 taagatgcaa gagttcgaat ctcttagcaa ccattatttt tttcctcaac ataacgagaa
     3061 cacacagggg cgctatcgca cagaatcaaa ttcgatgact ggaaattttt tgttaatttc
     3121 agaggtcgcc tgacgcatat acctttttca actgaaaaat tgggagaaaa aggaaaggtg
     3181 agagcgccgg aaccggcttt tcatatagaa tagagaagcg ttcatgacta aatgcttgca
     3241 tcacaatact tgaagttgac aatattattt aaggacctat tgttttttcc aataggtggt
     3301 tagcaatcgt cttactttct aacttttctt accttttaca tttcagcaat atatatatat
     3361 atatttcaag gatataccat tctaatgtct gcccctaaga agatcgtcgt tttgccaggt
     3421 gaccacgttg gtcaagaaat cacagccgaa gccattaagg ttcttaaagc tatttctgat
     3481 gttcgttcca atgtcaagtt cgatttcgaa aatcatttaa ttggtggtgc tgctatcgat
     3541 gctacaggtg ttccacttcc agatgaggcg ctggaagcct ccaagaaggc tgatgccgtt
     3601 ttgttaggtg ctgtgggtgg tcctaaatgg ggtaccggta gtgttagacc tgaacaaggt
     3661 ttactaaaaa tccgtaaaga acttcaattg tacgccaact taagaccatg taactttgca
     3721 tccgactctc ttttagactt atctccaatc aagccacaat ttgctaaagg tactgacttc
     3781 gttgttgtca gagaattagt gggaggtatt tactttggta agagaaagga agacgatggt
     3841 gatggtgtcg cttgggatag tgaacaatac accgttccag aagtgcaaag aatcacaaga
     3901 atggccgctt tcatggccct acaacatgag ccaccattgc ctatttggtc cttggataaa
     3961 gctaatgttt tggcctcttc aagattatgg agaaaaactg tggaggaaac catcaagaac
     4021 gaattcccta cattgaaggt tcaacatcaa ttgattgatt ctgccgccat gatcctagtt
     4081 aagaacccaa cccacctaaa tggtattata atcaccagca acatgtttgg tgatatcatc
     4141 tccgatgaag cctccgttat cccaggttcc ttgggtttgt tgccatctgc gtccttggcc
     4201 tctttgccag acaagaacac cgcatttggt ttgtacgaac catgccacgg ttctgctcca
     4261 gatttgccaa agaataaggt caaccctatc gccactatct tgtctgctgc aatgatgttg
     4321 aaattgtcat tgaacttgcc tgaagaaggt aaggccattg aagatgcagt taaaaaggtt
     4381 ttggatgcag gtatcagaac tggtgattta ggtggttcca acagtaccac cgaagtcggt
     4441 gatgctgtcg ccgaagaagt taagaaaatc cttgcttaaa aagattctct ttttttatga
     4501 tatttgtaca taaactttat aaatgaaatt cataatagaa acgacacgaa attacaaaat
     4561 ggaatatgtt catagggtag acgaaactat atacgcaatc tacatacatt tatcaagaag
     4621 gagaaaaagg aggatgtaaa ggaatacagg taagcaaatt gatactaatg gctcaacgtg
     4681 ataaggaaaa agaattgcac tttaacatta atattgacaa ggaggagggc accacacaaa
     4741 aagttaggtg taacagaaaa tcatgaaact atgattccta atttatatat tggaggattt
     4801 tctctaaaaa aaaaaaaata caacaaataa aaaacactca atgacctgac catttgatgg
     4861 agtttaagtc aataccttct tgaaccattt cccataatgg tgaaagttcc ctcaagaatt
     4921 ttactctgtc agaaacggcc ttaacgacgt agtcgatatg gtgcactctc agtacaatct
     4981 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     5041 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     5101 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     5161 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttaggac ggatcgcttg
     5221 cctgtaactt acacgcgcct cgtatctttt aatgatggaa taatttggga atttactctg
     5281 tgtttattta tttttatgtt ttgtatttgg attttagaaa gtaaataaag aaggtagaag
     5341 agttacggaa tgaagaaaaa aaaataaaca aaggtttaaa aaatttcaac aaaaagcgta
     5401 ctttacatat atatttatta gacaagaaaa gcagattaaa tagatataca ttcgattaac
     5461 gataagtaaa atgtaaaatc acaggatttt cgtgtgtggt cttctacaca gacaagatga
     5521 aacaattcgg cattaatacc tgagagcagg aagagcaaga taaaaggtag tatttgttgg
     5581 cgatccccct agagtctttt acatcttcgg aaaacaaaaa ctattttttc tttaatttct
     5641 ttttttactt tctattttta atttatatat ttatattaaa aaatttaaat tataattatt
     5701 tttatagcac gtgatgaaaa ggacccaggt ggcacttttc ggggaaatgt gcgcggaacc
     5761 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     5821 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     5881 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     5941 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     6001 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     6061 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     6121 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     6181 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     6241 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     6301 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     6361 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     6421 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     6481 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     6541 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     6601 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     6661 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     6721 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     6781 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     6841 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6901 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6961 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     7021 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     7081 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     7141 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     7201 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     7261 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     7321 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     7381 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     7441 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     7501 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     7561 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     7621 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     7681 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     7741 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     7801 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     7861 acaggaaaca gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagg
//
