LOCUS       sequence-256064-        8852 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 8852)
  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 8852)
  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 S389,398 D
FEATURES             Location/Qualifiers
     source          1..8852
                     /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     complement(2636..2652)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2637..2653)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      2655..2902
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2671..2689)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2920..2939)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2921..2939)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2949..2966)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2949..2965)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2958..2980)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3106..3561
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3193..3212)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3403..3424
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3862..4269
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             4270..5364
                     /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(5858..5877)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5977..5999
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6037..6055)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    6097..6600
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        6637..6741
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6742..6810,6811..7602)
                     /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(6960..6979)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7773..8361
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8262..8281
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8515..8532
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8649..8670
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8685..8690,8691..8708,8709..8715)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8723..8739
                     /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     8728..8750
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8747..8763
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8747..8763
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     8782..8802
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        8784..8802
                     /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 tctagagcat
      361 gtccaagggt gaagagctat ttactggggt tgtacccatt ttggtagaac tggacggaga
      421 tgtaaacgga cataaattct ctgttagagg tgagggcgaa ggcgatgcca ccaatggtaa
      481 attgactctg aagtttatat gcactacggg taaattacct gttccttggc caaccctagt
      541 aacaactttg acatatggtg ttcaatgttt ctcaagatac ccagaccata tgaaaaggca
      601 tgatttcttt aaaagtgcta tgccagaagg ctacgtgcaa gagagaacta tctcctttaa
      661 ggatgacggt acgtataaaa cacgagcaga agtgaaattc gaaggggata cactagttaa
      721 tcgcatcgaa ttaaagggta tagactttaa ggaagatggt aatattctcg gccataaact
      781 tgagtataat ttcaactcgc ataatgtgta cattacagct gacaaacaaa agaacggaat
      841 taaagcgaat tttaaaatca ggcacaacgt cgaagatggg tctgttcaac ttgccgatca
      901 ttatcagcaa aacaccccta ttggtgatgg tccagtcttg ttacctgata atcactactt
      961 aagcacacag tcgagattat ccaaagatcc aaacgaaaag agagaccaca tggtcttgtt
     1021 agaatttgtt actgctgctg gtattaccca tggtatggat gaattgtaca aaggcggcgg
     1081 cagcgccgcc gccggcggcg gcagccgcgg atccgcgatg agtaacgatg aaggagaaac
     1141 atttgccacg gaacagacta ctcaacaagt ctttcagaag ctaggtagca acatggaaaa
     1201 tcgagtttgt tttgattgtg gtaacaagaa tcctacttgg acttcggtac cttttggtgt
     1261 gatgctgtgt attcaatgct ctgctgtgca tagaaacatg ggtgttcata tcacctttgt
     1321 aaagtcttct acattagata aatggacaat taacaattta agaaggttta agttgggcgg
     1381 gaaccataag gctcgtgatt ttttcttaaa gaacaatggg aaacagctac taaatacagc
     1441 aaatgttgac gcgaaaacaa aatatacgag ccctgttgct aagaaatata agatacatct
     1501 cgacaagaag gttcaaaagg atatggagtt atacccatca gaattagtct tgaacggtca
     1561 agattcttcg gactcaccgt tggacaccga ttcagacgct tccaggtcga cttccaaaga
     1621 aaatagtgtt gacgattttt tctctaattg gcaaaaacca agctccaatt ccagttctaa
     1681 actcaatgta aatacaggct ctctcgcccc aaaaaacaac accacaggca gcacacccaa
     1741 gaccactgtc actaaaacga gaagttccat cttaacggct agcagaaaga aaccggtatt
     1801 aaactcgcaa gataaaaaga aacattcaat tttgtcttcg tcaagaaagc ccactagact
     1861 aaccgcaaag aaagtcgaca aatcacaggc agaagacttg tttgatcaat ttaaaaaaga
     1921 ggctcaacaa gaaaaggagg acgagttcac taattcgtct tctagcacca aaattagaca
     1981 aaatgattac gattcccaat ttatgaataa cagtaaaggc aacaataata acagtataga
     2041 tgatattaat acccagcctg atgaattcaa tgatttttta aatgacactt caaacagctt
     2101 cgacacaact agaaaagaac agcaagatac tttaacaccc aagtttgcca aattgggatt
     2161 tggtatgacc atgaatgacg ccaatgattt ggcaaagcaa caaaaagaat ctcagaaaat
     2221 agctcaggga ccaagatata cgggcagaat cgcggaaagg tacggcacac aaaaagctat
     2281 agattctgat caattatttg gtcgtggcga ttttgatgaa gcagcaaaca gggaagcgca
     2341 tgacaagttg aaaacatttg acaatgctac atcaatttcc tcttcttcat actttggaga
     2401 agataaagaa gtggatgaat ttggtaatcc catcaatagt agtggaagtg gagcagggaa
     2461 ttttgatggc cgtaattcta ataacggttt catcgatttt aatgcctccg cagatgatga
     2521 attgcagatg ttgagagacg ttgtagaaca gggtgctgaa aaactcggta gttatttaag
     2581 ggactactta agaaaataat ccccccgggc tgcaggaatt cgatatcaag cttatcgata
     2641 ccgtcgacct cgagtcatgt aattagttat gtcacgctta cattcacgcc ctccccccac
     2701 atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt
     2761 ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct
     2821 gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg
     2881 acgctcgaag gctttaattt gcggccggta cccaattcgc cctatagtga gtcgtattac
     2941 gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     3001 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     3061 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg
     3121 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     3181 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     3241 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     3301 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     3361 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     3421 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     3481 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     3541 aaatattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3601 ttcacaccgc atatcgaccg gtcgaggaga acttctagta tatctacata cctaatatta
     3661 ttgccttatt aaaaatggaa tcccaacaat tacatcaaaa tccacattct cttcaaaatc
     3721 aattgtcctg tacttccttg ttcatgtgtg ttcaaaaacg ttatatttat aggataatta
     3781 tactctattt ctcaacaagt aattggttgt ttggccgagc ggtctaaggc gcctgattca
     3841 agaaatatct tgaccgcagt taactgtggg aatactcagg tatcgtaaga tgcaagagtt
     3901 cgaatctctt agcaaccatt atttttttcc tcaacataac gagaacacac aggggcgcta
     3961 tcgcacagaa tcaaattcga tgactggaaa ttttttgtta atttcagagg tcgcctgacg
     4021 catatacctt tttcaactga aaaattggga gaaaaaggaa aggtgagagc gccggaaccg
     4081 gcttttcata tagaatagag aagcgttcat gactaaatgc ttgcatcaca atacttgaag
     4141 ttgacaatat tatttaagga cctattgttt tttccaatag gtggttagca atcgtcttac
     4201 tttctaactt ttcttacctt ttacatttca gcaatatata tatatatatt tcaaggatat
     4261 accattctaa tgtctgcccc taagaagatc gtcgttttgc caggtgacca cgttggtcaa
     4321 gaaatcacag ccgaagccat taaggttctt aaagctattt ctgatgttcg ttccaatgtc
     4381 aagttcgatt tcgaaaatca tttaattggt ggtgctgcta tcgatgctac aggtgttcca
     4441 cttccagatg aggcgctgga agcctccaag aaggctgatg ccgttttgtt aggtgctgtg
     4501 ggtggtccta aatggggtac cggtagtgtt agacctgaac aaggtttact aaaaatccgt
     4561 aaagaacttc aattgtacgc caacttaaga ccatgtaact ttgcatccga ctctctttta
     4621 gacttatctc caatcaagcc acaatttgct aaaggtactg acttcgttgt tgtcagagaa
     4681 ttagtgggag gtatttactt tggtaagaga aaggaagacg atggtgatgg tgtcgcttgg
     4741 gatagtgaac aatacaccgt tccagaagtg caaagaatca caagaatggc cgctttcatg
     4801 gccctacaac atgagccacc attgcctatt tggtccttgg ataaagctaa tgttttggcc
     4861 tcttcaagat tatggagaaa aactgtggag gaaaccatca agaacgaatt ccctacattg
     4921 aaggttcaac atcaattgat tgattctgcc gccatgatcc tagttaagaa cccaacccac
     4981 ctaaatggta ttataatcac cagcaacatg tttggtgata tcatctccga tgaagcctcc
     5041 gttatcccag gttccttggg tttgttgcca tctgcgtcct tggcctcttt gccagacaag
     5101 aacaccgcat ttggtttgta cgaaccatgc cacggttctg ctccagattt gccaaagaat
     5161 aaggtcaacc ctatcgccac tatcttgtct gctgcaatga tgttgaaatt gtcattgaac
     5221 ttgcctgaag aaggtaaggc cattgaagat gcagttaaaa aggttttgga tgcaggtatc
     5281 agaactggtg atttaggtgg ttccaacagt accaccgaag tcggtgatgc tgtcgccgaa
     5341 gaagttaaga aaatccttgc ttaaaaagat tctctttttt tatgatattt gtacataaac
     5401 tttataaatg aaattcataa tagaaacgac acgaaattac aaaatggaat atgttcatag
     5461 ggtagacgaa actatatacg caatctacat acatttatca agaaggagaa aaaggaggat
     5521 gtaaaggaat acaggtaagc aaattgatac taatggctca acgtgataag gaaaaagaat
     5581 tgcactttaa cattaatatt gacaaggagg agggcaccac acaaaaagtt aggtgtaaca
     5641 gaaaatcatg aaactatgat tcctaattta tatattggag gattttctct aaaaaaaaaa
     5701 aaatacaaca aataaaaaac actcaatgac ctgaccattt gatggagttt aagtcaatac
     5761 cttcttgaac catttcccat aatggtgaaa gttccctcaa gaattttact ctgtcagaaa
     5821 cggccttaac gacgtagtcg atatggtgca ctctcagtac aatctgctct gatgccgcat
     5881 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     5941 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     6001 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     6061 aggttaatgt catgataata atggtttctt aggacggatc gcttgcctgt aacttacacg
     6121 cgcctcgtat cttttaatga tggaataatt tgggaattta ctctgtgttt atttattttt
     6181 atgttttgta tttggatttt agaaagtaaa taaagaaggt agaagagtta cggaatgaag
     6241 aaaaaaaaat aaacaaaggt ttaaaaaatt tcaacaaaaa gcgtacttta catatatatt
     6301 tattagacaa gaaaagcaga ttaaatagat atacattcga ttaacgataa gtaaaatgta
     6361 aaatcacagg attttcgtgt gtggtcttct acacagacaa gatgaaacaa ttcggcatta
     6421 atacctgaga gcaggaagag caagataaaa ggtagtattt gttggcgatc cccctagagt
     6481 cttttacatc ttcggaaaac aaaaactatt ttttctttaa tttctttttt tactttctat
     6541 ttttaattta tatatttata ttaaaaaatt taaattataa ttatttttat agcacgtgat
     6601 gaaaaggacc caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     6661 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     6721 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     6781 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     6841 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     6901 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     6961 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     7021 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     7081 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     7141 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     7201 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     7261 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     7321 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     7381 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     7441 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     7501 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     7561 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     7621 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     7681 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     7741 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     7801 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     7861 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     7921 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     7981 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     8041 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     8101 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     8161 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     8221 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     8281 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     8341 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     8401 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     8461 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     8521 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
     8581 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     8641 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     8701 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     8761 gaccatgatt acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctc
     8821 cggatgcaag ggttcgaatc ccttagctct ca
//
