LOCUS       sequence-256191-        8498 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 8498)
  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 8498)
  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 GFP-Glo3 Delta-GRM-Delta-C
FEATURES             Location/Qualifiers
     source          1..8498
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1931..1947)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1932..1948)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      1950..2197
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1966..1984)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2215..2234)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2216..2234)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2244..2261)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2244..2260)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2253..2275)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2401..2856
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2488..2507)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2698..2719
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3157..3564
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             3565..4659
                     /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(5153..5172)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5272..5294
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5332..5350)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    5392..5895
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5932..6036
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6037..6105,6106..6897)
                     /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(6255..6274)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7068..7656
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7557..7576
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7810..7827
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7944..7965
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7980..7985,7986..8003,8004..8010)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8018..8034
                     /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     8023..8045
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8042..8058
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8042..8058
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     8077..8097
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        8079..8097
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        8148..8497
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /label=MET17 promoter
                     /note="expression is repressed by methionine"
ORIGIN
        1 ctagagcatg tccaagggtg aagagctatt tactggggtt gtacccattt tggtagaact
       61 ggacggagat gtaaacggac ataaattctc tgttagaggt gagggcgaag gcgatgccac
      121 caatggtaaa ttgactctga agtttatatg cactacgggt aaattacctg ttccttggcc
      181 aaccctagta acaactttga catatggtgt tcaatgtttc tcaagatacc cagaccatat
      241 gaaaaggcat gatttcttta aaagtgctat gccagaaggc tacgtgcaag agagaactat
      301 ctcctttaag gatgacggta cgtataaaac acgagcagaa gtgaaattcg aaggggatac
      361 actagttaat cgcatcgaat taaagggtat agactttaag gaagatggta atattctcgg
      421 ccataaactt gagtataatt tcaactcgca taatgtgtac attacagctg acaaacaaaa
      481 gaacggaatt aaagcgaatt ttaaaatcag gcacaacgtc gaagatgggt ctgttcaact
      541 tgccgatcat tatcagcaaa acacccctat tggtgatggt ccagtcttgt tacctgataa
      601 tcactactta agcacacagt cgagattatc caaagatcca aacgaaaaga gagaccacat
      661 ggtcttgtta gaatttgtta ctgctgctgg tattacccat ggtatggatg aattgtacaa
      721 aggcggcggc agcgccgccg ccggcggcgg cagccgcgga tccgcgatga gtaacgatga
      781 aggagaaaca tttgccacgg aacagactac tcaacaagtc tttcagaagc taggtagcaa
      841 catggaaaat cgagtttgtt ttgattgtgg taacaagaat cctacttgga cttcggtacc
      901 ttttggtgtg atgctgtgta ttcaatgctc tgctgtgcat agaaacatgg gtgttcatat
      961 cacctttgta aagtcttcta cattagataa atggacaatt aacaatttaa gaaggtttaa
     1021 gttgggcggg aaccataagg ctcgtgattt tttcttaaag aacaatggga aacagctact
     1081 aaatacagca aatgttgacg cgaaaacaaa atatacgagc cctgttgcta agaaatataa
     1141 gatacatctc gacaagaagg ttcaaaagga tatggagtta tacccatcag aattagtctt
     1201 gaacggtcaa gattcttcgg actcaccgtt ggacaccgat tcagacgctt ccaggtcgac
     1261 ttccaaagaa aatagtgttg acgatttttt ctctaattgg caaaaaccaa gctccaattc
     1321 cagttctaaa ctcaatgtaa atacaggctc tctcgcccca aaaaacaaca ccacaggcag
     1381 cacacccaag accactgtca ctaaaacgag aagttccatc ttaacggcta gcagaaagaa
     1441 accggtatta aactcgcaag ataaaaagaa acattcaatt ttgtcttcgt caagaaagcc
     1501 cactagacta accgcaaaga aagtcgacaa atcacaggca gaagacttgt ttgatcaatt
     1561 taaaaaagag gctcaacaag aaaaggagga cgagttcact aattcgtctt ctagcaccaa
     1621 aattagacaa aatgattacg attcccaatt tatgaataac agtaaaggca acaataataa
     1681 cagtatagat gatattaata cccagcctga tgaattcaat gattttttaa atgacacttc
     1741 aaacagcttc gacacaacta gaaaagaaca gcaagatact ttaacaccca agtttgccaa
     1801 attgggattt ggtatgacca tgaatgacgc caatgatttg gcaaagcaac aaaaagaatc
     1861 tcagaaaata gctcagggac caagatatac gtaatccccc cgggctgcag gaattcgata
     1921 tcaagcttat cgataccgtc gacctcgagt catgtaatta gttatgtcac gcttacattc
     1981 acgccctccc cccacatccg ctctaaccga aaaggaagga gttagacaac ctgaagtcta
     2041 ggtccctatt tattttttta tagttatgtt agtattaaga acgttattta tatttcaaat
     2101 ttttcttttt tttctgtaca gacgcgtgta cgcatgtaac attatactga aaaccttgct
     2161 tgagaaggtt ttgggacgct cgaaggcttt aatttgcggc cggtacccaa ttcgccctat
     2221 agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     2281 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     2341 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     2401 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     2461 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     2521 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     2581 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     2641 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     2701 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     2761 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     2821 acgcgaattt taacaaaata ttaacgctta caatttcctg atgcggtatt ttctccttac
     2881 gcatctgtgc ggtatttcac accgcatatc gaccggtcga ggagaacttc tagtatatct
     2941 acatacctaa tattattgcc ttattaaaaa tggaatccca acaattacat caaaatccac
     3001 attctcttca aaatcaattg tcctgtactt ccttgttcat gtgtgttcaa aaacgttata
     3061 tttataggat aattatactc tatttctcaa caagtaattg gttgtttggc cgagcggtct
     3121 aaggcgcctg attcaagaaa tatcttgacc gcagttaact gtgggaatac tcaggtatcg
     3181 taagatgcaa gagttcgaat ctcttagcaa ccattatttt tttcctcaac ataacgagaa
     3241 cacacagggg cgctatcgca cagaatcaaa ttcgatgact ggaaattttt tgttaatttc
     3301 agaggtcgcc tgacgcatat acctttttca actgaaaaat tgggagaaaa aggaaaggtg
     3361 agagcgccgg aaccggcttt tcatatagaa tagagaagcg ttcatgacta aatgcttgca
     3421 tcacaatact tgaagttgac aatattattt aaggacctat tgttttttcc aataggtggt
     3481 tagcaatcgt cttactttct aacttttctt accttttaca tttcagcaat atatatatat
     3541 atatttcaag gatataccat tctaatgtct gcccctaaga agatcgtcgt tttgccaggt
     3601 gaccacgttg gtcaagaaat cacagccgaa gccattaagg ttcttaaagc tatttctgat
     3661 gttcgttcca atgtcaagtt cgatttcgaa aatcatttaa ttggtggtgc tgctatcgat
     3721 gctacaggtg ttccacttcc agatgaggcg ctggaagcct ccaagaaggc tgatgccgtt
     3781 ttgttaggtg ctgtgggtgg tcctaaatgg ggtaccggta gtgttagacc tgaacaaggt
     3841 ttactaaaaa tccgtaaaga acttcaattg tacgccaact taagaccatg taactttgca
     3901 tccgactctc ttttagactt atctccaatc aagccacaat ttgctaaagg tactgacttc
     3961 gttgttgtca gagaattagt gggaggtatt tactttggta agagaaagga agacgatggt
     4021 gatggtgtcg cttgggatag tgaacaatac accgttccag aagtgcaaag aatcacaaga
     4081 atggccgctt tcatggccct acaacatgag ccaccattgc ctatttggtc cttggataaa
     4141 gctaatgttt tggcctcttc aagattatgg agaaaaactg tggaggaaac catcaagaac
     4201 gaattcccta cattgaaggt tcaacatcaa ttgattgatt ctgccgccat gatcctagtt
     4261 aagaacccaa cccacctaaa tggtattata atcaccagca acatgtttgg tgatatcatc
     4321 tccgatgaag cctccgttat cccaggttcc ttgggtttgt tgccatctgc gtccttggcc
     4381 tctttgccag acaagaacac cgcatttggt ttgtacgaac catgccacgg ttctgctcca
     4441 gatttgccaa agaataaggt caaccctatc gccactatct tgtctgctgc aatgatgttg
     4501 aaattgtcat tgaacttgcc tgaagaaggt aaggccattg aagatgcagt taaaaaggtt
     4561 ttggatgcag gtatcagaac tggtgattta ggtggttcca acagtaccac cgaagtcggt
     4621 gatgctgtcg ccgaagaagt taagaaaatc cttgcttaaa aagattctct ttttttatga
     4681 tatttgtaca taaactttat aaatgaaatt cataatagaa acgacacgaa attacaaaat
     4741 ggaatatgtt catagggtag acgaaactat atacgcaatc tacatacatt tatcaagaag
     4801 gagaaaaagg aggatgtaaa ggaatacagg taagcaaatt gatactaatg gctcaacgtg
     4861 ataaggaaaa agaattgcac tttaacatta atattgacaa ggaggagggc accacacaaa
     4921 aagttaggtg taacagaaaa tcatgaaact atgattccta atttatatat tggaggattt
     4981 tctctaaaaa aaaaaaaata caacaaataa aaaacactca atgacctgac catttgatgg
     5041 agtttaagtc aataccttct tgaaccattt cccataatgg tgaaagttcc ctcaagaatt
     5101 ttactctgtc agaaacggcc ttaacgacgt agtcgatatg gtgcactctc agtacaatct
     5161 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     5221 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     5281 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     5341 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttaggac ggatcgcttg
     5401 cctgtaactt acacgcgcct cgtatctttt aatgatggaa taatttggga atttactctg
     5461 tgtttattta tttttatgtt ttgtatttgg attttagaaa gtaaataaag aaggtagaag
     5521 agttacggaa tgaagaaaaa aaaataaaca aaggtttaaa aaatttcaac aaaaagcgta
     5581 ctttacatat atatttatta gacaagaaaa gcagattaaa tagatataca ttcgattaac
     5641 gataagtaaa atgtaaaatc acaggatttt cgtgtgtggt cttctacaca gacaagatga
     5701 aacaattcgg cattaatacc tgagagcagg aagagcaaga taaaaggtag tatttgttgg
     5761 cgatccccct agagtctttt acatcttcgg aaaacaaaaa ctattttttc tttaatttct
     5821 ttttttactt tctattttta atttatatat ttatattaaa aaatttaaat tataattatt
     5881 tttatagcac gtgatgaaaa ggacccaggt ggcacttttc ggggaaatgt gcgcggaacc
     5941 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     6001 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     6061 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     6121 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     6181 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     6241 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     6301 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     6361 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     6421 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     6481 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     6541 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     6601 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     6661 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     6721 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     6781 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     6841 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     6901 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     6961 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     7021 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     7081 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     7141 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     7201 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     7261 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     7321 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     7381 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     7441 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     7501 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     7561 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     7621 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     7681 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     7741 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     7801 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     7861 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     7921 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     7981 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     8041 acaggaaaca gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa
     8101 caaaagctgg agctccggat gcaagggttc gaatccctta gctctcatta ttttttgctt
     8161 tttctcttga ggtcacatga tcgcaaaatg gcaaatggca cgtgaagctg tcgatattgg
     8221 ggaactgtgg tggttggcaa atgactaatt aagttagtca aggcgccatc ctcatgaaaa
     8281 ctgtgtaaca taataaccga agtgtcgaaa aggtggcacc ttgtccaatt gaacacgctc
     8341 gatgaaaaaa ataagatata tataaggtta agtaaagcgt ctgttagaaa ggaagttttt
     8401 cctttttctt gctctcttgt cttttcatct actatttcct tcgtgtaata cagggtcgtc
     8461 agatacatag atacaattct attaccccca tccatact
//
