LOCUS       sequence-256370-        7752 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression in S. cerevisiae.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7752)
  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 7752)
  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 Met25 Glo3 Delta-GRM-Delta-C
FEATURES             Location/Qualifiers
     source          1..7752
                     /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(1536..1552)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1537..1553)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      1555..1802
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1571..1589)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(1820..1839)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1821..1839)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1849..1866)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1849..1865)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1858..1880)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2006..2461
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2093..2112)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2303..2324
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2762..3169
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             3170..4264
                     /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(4758..4777)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4877..4899
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4937..4955)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4997..5500
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5537..5641
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5642..5710,5711..6502)
                     /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(5860..5879)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6673..7261
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7162..7181
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7415..7432
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7549..7570
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7585..7590,7591..7608,7609..7615)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7623..7639
                     /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     7628..7650
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7647..7663
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7647..7663
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7682..7702
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7684..7702
                     /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 gatgagtaac gatgaaggag aaacatttgc cacggaacag actactcaac
      421 aagtctttca gaagctaggt agcaacatgg aaaatcgagt ttgttttgat tgtggtaaca
      481 agaatcctac ttggacttcg gtaccttttg gtgtgatgct gtgtattcaa tgctctgctg
      541 tgcatagaaa catgggtgtt catatcacct ttgtaaagtc ttctacatta gataaatgga
      601 caattaacaa tttaagaagg tttaagttgg gcgggaacca taaggctcgt gattttttct
      661 taaagaacaa tgggaaacag ctactaaata cagcaaatgt tgacgcgaaa acaaaatata
      721 cgagccctgt tgctaagaaa tataagatac atctcgacaa gaaggttcaa aaggatatgg
      781 agttataccc atcagaatta gtcttgaacg gtcaagattc ttcggactca ccgttggaca
      841 ccgattcaga cgcttccagg tcgacttcca aagaaaatag tgttgacgat tttttctcta
      901 attggcaaaa accaagctcc aattccagtt ctaaactcaa tgtaaataca ggctctctcg
      961 ccccaaaaaa caacaccaca ggcagcacac ccaagaccac tgtcactaaa acgagaagtt
     1021 ccatcttaac ggctagcaga aagaaaccgg tattaaactc gcaagataaa aagaaacatt
     1081 caattttgtc ttcgtcaaga aagcccacta gactaaccgc aaagaaagtc gacaaatcac
     1141 aggcagaaga cttgtttgat caatttaaaa aagaggctca acaagaaaag gaggacgagt
     1201 tcactaattc gtcttctagc accaaaatta gacaaaatga ttacgattcc caatttatga
     1261 ataacagtaa aggcaacaat aataacagta tagatgatat taatacccag cctgatgaat
     1321 tcaatgattt tttaaatgac acttcaaaca gcttcgacac aactagaaaa gaacagcaag
     1381 atactttaac acccaagttt gccaaattgg gatttggtat gaccatgaat gacgccaatg
     1441 atttggcaaa gcaacaaaaa gaatctcaga aaatagctca gggaccaaga tatacgtaat
     1501 ccccccgggc tgcaggaatt cgatatcaag cttatcgata ccgtcgacct cgagtcatgt
     1561 aattagttat gtcacgctta cattcacgcc ctccccccac atccgctcta accgaaaagg
     1621 aaggagttag acaacctgaa gtctaggtcc ctatttattt ttttatagtt atgttagtat
     1681 taagaacgtt atttatattt caaatttttc ttttttttct gtacagacgc gtgtacgcat
     1741 gtaacattat actgaaaacc ttgcttgaga aggttttggg acgctcgaag gctttaattt
     1801 gcggccggta cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt
     1861 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     1921 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     1981 gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2041 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2101 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2161 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2221 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2281 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     2341 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     2401 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     2461 tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatcgaccg
     2521 gtcgaggaga acttctagta tatctacata cctaatatta ttgccttatt aaaaatggaa
     2581 tcccaacaat tacatcaaaa tccacattct cttcaaaatc aattgtcctg tacttccttg
     2641 ttcatgtgtg ttcaaaaacg ttatatttat aggataatta tactctattt ctcaacaagt
     2701 aattggttgt ttggccgagc ggtctaaggc gcctgattca agaaatatct tgaccgcagt
     2761 taactgtggg aatactcagg tatcgtaaga tgcaagagtt cgaatctctt agcaaccatt
     2821 atttttttcc tcaacataac gagaacacac aggggcgcta tcgcacagaa tcaaattcga
     2881 tgactggaaa ttttttgtta atttcagagg tcgcctgacg catatacctt tttcaactga
     2941 aaaattggga gaaaaaggaa aggtgagagc gccggaaccg gcttttcata tagaatagag
     3001 aagcgttcat gactaaatgc ttgcatcaca atacttgaag ttgacaatat tatttaagga
     3061 cctattgttt tttccaatag gtggttagca atcgtcttac tttctaactt ttcttacctt
     3121 ttacatttca gcaatatata tatatatatt tcaaggatat accattctaa tgtctgcccc
     3181 taagaagatc gtcgttttgc caggtgacca cgttggtcaa gaaatcacag ccgaagccat
     3241 taaggttctt aaagctattt ctgatgttcg ttccaatgtc aagttcgatt tcgaaaatca
     3301 tttaattggt ggtgctgcta tcgatgctac aggtgttcca cttccagatg aggcgctgga
     3361 agcctccaag aaggctgatg ccgttttgtt aggtgctgtg ggtggtccta aatggggtac
     3421 cggtagtgtt agacctgaac aaggtttact aaaaatccgt aaagaacttc aattgtacgc
     3481 caacttaaga ccatgtaact ttgcatccga ctctctttta gacttatctc caatcaagcc
     3541 acaatttgct aaaggtactg acttcgttgt tgtcagagaa ttagtgggag gtatttactt
     3601 tggtaagaga aaggaagacg atggtgatgg tgtcgcttgg gatagtgaac aatacaccgt
     3661 tccagaagtg caaagaatca caagaatggc cgctttcatg gccctacaac atgagccacc
     3721 attgcctatt tggtccttgg ataaagctaa tgttttggcc tcttcaagat tatggagaaa
     3781 aactgtggag gaaaccatca agaacgaatt ccctacattg aaggttcaac atcaattgat
     3841 tgattctgcc gccatgatcc tagttaagaa cccaacccac ctaaatggta ttataatcac
     3901 cagcaacatg tttggtgata tcatctccga tgaagcctcc gttatcccag gttccttggg
     3961 tttgttgcca tctgcgtcct tggcctcttt gccagacaag aacaccgcat ttggtttgta
     4021 cgaaccatgc cacggttctg ctccagattt gccaaagaat aaggtcaacc ctatcgccac
     4081 tatcttgtct gctgcaatga tgttgaaatt gtcattgaac ttgcctgaag aaggtaaggc
     4141 cattgaagat gcagttaaaa aggttttgga tgcaggtatc agaactggtg atttaggtgg
     4201 ttccaacagt accaccgaag tcggtgatgc tgtcgccgaa gaagttaaga aaatccttgc
     4261 ttaaaaagat tctctttttt tatgatattt gtacataaac tttataaatg aaattcataa
     4321 tagaaacgac acgaaattac aaaatggaat atgttcatag ggtagacgaa actatatacg
     4381 caatctacat acatttatca agaaggagaa aaaggaggat gtaaaggaat acaggtaagc
     4441 aaattgatac taatggctca acgtgataag gaaaaagaat tgcactttaa cattaatatt
     4501 gacaaggagg agggcaccac acaaaaagtt aggtgtaaca gaaaatcatg aaactatgat
     4561 tcctaattta tatattggag gattttctct aaaaaaaaaa aaatacaaca aataaaaaac
     4621 actcaatgac ctgaccattt gatggagttt aagtcaatac cttcttgaac catttcccat
     4681 aatggtgaaa gttccctcaa gaattttact ctgtcagaaa cggccttaac gacgtagtcg
     4741 atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     4801 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     4861 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     4921 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     4981 atggtttctt aggacggatc gcttgcctgt aacttacacg cgcctcgtat cttttaatga
     5041 tggaataatt tgggaattta ctctgtgttt atttattttt atgttttgta tttggatttt
     5101 agaaagtaaa taaagaaggt agaagagtta cggaatgaag aaaaaaaaat aaacaaaggt
     5161 ttaaaaaatt tcaacaaaaa gcgtacttta catatatatt tattagacaa gaaaagcaga
     5221 ttaaatagat atacattcga ttaacgataa gtaaaatgta aaatcacagg attttcgtgt
     5281 gtggtcttct acacagacaa gatgaaacaa ttcggcatta atacctgaga gcaggaagag
     5341 caagataaaa ggtagtattt gttggcgatc cccctagagt cttttacatc ttcggaaaac
     5401 aaaaactatt ttttctttaa tttctttttt tactttctat ttttaattta tatatttata
     5461 ttaaaaaatt taaattataa ttatttttat agcacgtgat gaaaaggacc caggtggcac
     5521 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5581 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     5641 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     5701 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5761 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5821 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5881 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     5941 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     6001 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     6061 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     6121 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     6181 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     6241 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     6301 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     6361 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     6421 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     6481 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     6541 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     6601 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6661 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6721 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6781 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     6841 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6901 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6961 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     7021 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     7081 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     7141 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     7201 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     7261 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     7321 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     7381 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     7441 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     7501 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     7561 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     7621 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
     7681 cgcaattaac cctcactaaa gggaacaaaa gctggagctc cggatgcaag ggttcgaatc
     7741 ccttagctct ca
//
