LOCUS       sequence-256104-        8105 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 8105)
  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 8105)
  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 S389,398 D
FEATURES             Location/Qualifiers
     source          1..8105
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1..17
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     36..56
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        38..56
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        107..456
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /label=MET17 promoter
                     /note="expression is repressed by methionine"
     primer_bind     457..473
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     457..473
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1995..2011)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1996..2012)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      2014..2261
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2030..2048)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2279..2298)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2280..2298)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2308..2325)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2308..2324)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2317..2339)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2465..2920
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2552..2571)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2762..2783
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3221..3628
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             3629..4723
                     /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(5217..5236)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5336..5358
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5396..5414)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    5456..5959
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5996..6100
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6101..6169,6170..6961)
                     /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(6319..6338)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7132..7720
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7621..7640
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7874..7891
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8008..8029
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8044..8049,8050..8067,8068..8074)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8082..8098
                     /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     join(8087..8105,1..4)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac
       61 aaaagctgga gctccggatg caagggttcg aatcccttag ctctcattat tttttgcttt
      121 ttctcttgag gtcacatgat cgcaaaatgg caaatggcac gtgaagctgt cgatattggg
      181 gaactgtggt ggttggcaaa tgactaatta agttagtcaa ggcgccatcc tcatgaaaac
      241 tgtgtaacat aataaccgaa gtgtcgaaaa ggtggcacct tgtccaattg aacacgctcg
      301 atgaaaaaaa taagatatat ataaggttaa gtaaagcgtc tgttagaaag gaagtttttc
      361 ctttttcttg ctctcttgtc ttttcatcta ctatttcctt cgtgtaatac agggtcgtca
      421 gatacataga tacaattcta ttacccccat ccatactcta gaactagtgg atccgcatga
      481 gtaacgatga aggagaaaca tttgccacgg aacagactac tcaacaagtt tttcagaagc
      541 taggtagcaa catggaaaat cgagtttgtt ttgattgtgg taacaagaat cctacttgga
      601 cttcggtacc ttttggtgtg atgctgtgta ttcaatgctc tgctgtgcat aagaacatgg
      661 gtgttcatat cacctttgta aagtcttcta cattagataa atggacaatt aacaatttaa
      721 gaaggtttaa gttgggtggg aaccataagg ctcgtgattt tttcttaaag aacaatggga
      781 aacagctact aaatacagca aatgttgacg cgaaaacaaa atatacgagc cctgttgcta
      841 agaaatataa gatacatctc gacaagaagg ttcaaaagga tatggagtta tacccatcag
      901 aattagtctt gaacggtcaa gattcttcgg actcaccgtt ggacaccgat tcagacgctt
      961 ccaggtcgac ttccaaagaa aatagtgttg acgacttttt ctctaattgg caaaaaccaa
     1021 gctccaattc cagttctaaa ctcaatgtaa atacaggctc tctcgcccca aaaaacaaca
     1081 ccacaggcag cacacccaag accactgtca ctaaaacgag aagttccatc ttaacggcta
     1141 gcagaaagaa accggtatta aactcgcaag ataaaaagaa acattcaatt ttgtcttcgt
     1201 caagaaagcc cactagacta accgcaaaga aagtcgacaa atcacaggca gaagacttgt
     1261 ttgatcaatt taaaaaagag gctcaacaag aaaaggagga cgagttcact aattcgtctt
     1321 ctagcaccaa aattagacaa aatgattacg attcccaatt tatgaataac agtaaaggca
     1381 acaataataa cagtatagat gatattaata cccagcctga tgaattcaat gattttttaa
     1441 atgacacttc aaacagcttc gacacaacta gaaaagaaca gcaagatact ttaacaccca
     1501 agtttgccaa attgggattt ggtatgacca tgaatgacgc caatgatttg gcaaagcaac
     1561 aaaaagaatc tcagaaaata gctcagggac caagatatac gggcagaatc gcggaaaggt
     1621 acggcacaca aaaagctata gattctgatc aattatttgg tcgtggcgat tttgatgaag
     1681 cagcaaacag ggaagcgcat gacaagttga aaacatttga caatgctaca tcaatttcct
     1741 cttcttcata ctttggagaa gataaagaag tggatgaatt tggtaatccc atcaatagta
     1801 gtggaagtgg agcagggaat tttgatggcc gtaattctaa taacggtttc atcgatttta
     1861 atgcctccgc agatgatgaa ttgcagatgt tgagagacgt tgtagaacag ggtgctgaaa
     1921 aactcggtag ttatttaagg gactacttaa gaaaataatc cccccgggct gcaggaattc
     1981 gatatcaagc ttatcgatac cgtcgacctc gagtcatgta attagttatg tcacgcttac
     2041 attcacgccc tccccccaca tccgctctaa ccgaaaagga aggagttaga caacctgaag
     2101 tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc
     2161 aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct
     2221 tgcttgagaa ggttttggga cgctcgaagg ctttaatttg cggccggtac ccaattcgcc
     2281 ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga
     2341 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     2401 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     2461 atggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2521 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2581 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2641 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     2701 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     2761 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     2821 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     2881 tttaacgcga attttaacaa aatattaacg cttacaattt cctgatgcgg tattttctcc
     2941 ttacgcatct gtgcggtatt tcacaccgca tatcgaccgg tcgaggagaa cttctagtat
     3001 atctacatac ctaatattat tgccttatta aaaatggaat cccaacaatt acatcaaaat
     3061 ccacattctc ttcaaaatca attgtcctgt acttccttgt tcatgtgtgt tcaaaaacgt
     3121 tatatttata ggataattat actctatttc tcaacaagta attggttgtt tggccgagcg
     3181 gtctaaggcg cctgattcaa gaaatatctt gaccgcagtt aactgtggga atactcaggt
     3241 atcgtaagat gcaagagttc gaatctctta gcaaccatta tttttttcct caacataacg
     3301 agaacacaca ggggcgctat cgcacagaat caaattcgat gactggaaat tttttgttaa
     3361 tttcagaggt cgcctgacgc atataccttt ttcaactgaa aaattgggag aaaaaggaaa
     3421 ggtgagagcg ccggaaccgg cttttcatat agaatagaga agcgttcatg actaaatgct
     3481 tgcatcacaa tacttgaagt tgacaatatt atttaaggac ctattgtttt ttccaatagg
     3541 tggttagcaa tcgtcttact ttctaacttt tcttaccttt tacatttcag caatatatat
     3601 atatatattt caaggatata ccattctaat gtctgcccct aagaagatcg tcgttttgcc
     3661 aggtgaccac gttggtcaag aaatcacagc cgaagccatt aaggttctta aagctatttc
     3721 tgatgttcgt tccaatgtca agttcgattt cgaaaatcat ttaattggtg gtgctgctat
     3781 cgatgctaca ggtgttccac ttccagatga ggcgctggaa gcctccaaga aggctgatgc
     3841 cgttttgtta ggtgctgtgg gtggtcctaa atggggtacc ggtagtgtta gacctgaaca
     3901 aggtttacta aaaatccgta aagaacttca attgtacgcc aacttaagac catgtaactt
     3961 tgcatccgac tctcttttag acttatctcc aatcaagcca caatttgcta aaggtactga
     4021 cttcgttgtt gtcagagaat tagtgggagg tatttacttt ggtaagagaa aggaagacga
     4081 tggtgatggt gtcgcttggg atagtgaaca atacaccgtt ccagaagtgc aaagaatcac
     4141 aagaatggcc gctttcatgg ccctacaaca tgagccacca ttgcctattt ggtccttgga
     4201 taaagctaat gttttggcct cttcaagatt atggagaaaa actgtggagg aaaccatcaa
     4261 gaacgaattc cctacattga aggttcaaca tcaattgatt gattctgccg ccatgatcct
     4321 agttaagaac ccaacccacc taaatggtat tataatcacc agcaacatgt ttggtgatat
     4381 catctccgat gaagcctccg ttatcccagg ttccttgggt ttgttgccat ctgcgtcctt
     4441 ggcctctttg ccagacaaga acaccgcatt tggtttgtac gaaccatgcc acggttctgc
     4501 tccagatttg ccaaagaata aggtcaaccc tatcgccact atcttgtctg ctgcaatgat
     4561 gttgaaattg tcattgaact tgcctgaaga aggtaaggcc attgaagatg cagttaaaaa
     4621 ggttttggat gcaggtatca gaactggtga tttaggtggt tccaacagta ccaccgaagt
     4681 cggtgatgct gtcgccgaag aagttaagaa aatccttgct taaaaagatt ctcttttttt
     4741 atgatatttg tacataaact ttataaatga aattcataat agaaacgaca cgaaattaca
     4801 aaatggaata tgttcatagg gtagacgaaa ctatatacgc aatctacata catttatcaa
     4861 gaaggagaaa aaggaggatg taaaggaata caggtaagca aattgatact aatggctcaa
     4921 cgtgataagg aaaaagaatt gcactttaac attaatattg acaaggagga gggcaccaca
     4981 caaaaagtta ggtgtaacag aaaatcatga aactatgatt cctaatttat atattggagg
     5041 attttctcta aaaaaaaaaa aatacaacaa ataaaaaaca ctcaatgacc tgaccatttg
     5101 atggagttta agtcaatacc ttcttgaacc atttcccata atggtgaaag ttccctcaag
     5161 aattttactc tgtcagaaac ggccttaacg acgtagtcga tatggtgcac tctcagtaca
     5221 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     5281 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     5341 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     5401 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta ggacggatcg
     5461 cttgcctgta acttacacgc gcctcgtatc ttttaatgat ggaataattt gggaatttac
     5521 tctgtgttta tttattttta tgttttgtat ttggatttta gaaagtaaat aaagaaggta
     5581 gaagagttac ggaatgaaga aaaaaaaata aacaaaggtt taaaaaattt caacaaaaag
     5641 cgtactttac atatatattt attagacaag aaaagcagat taaatagata tacattcgat
     5701 taacgataag taaaatgtaa aatcacagga ttttcgtgtg tggtcttcta cacagacaag
     5761 atgaaacaat tcggcattaa tacctgagag caggaagagc aagataaaag gtagtatttg
     5821 ttggcgatcc ccctagagtc ttttacatct tcggaaaaca aaaactattt tttctttaat
     5881 ttcttttttt actttctatt tttaatttat atatttatat taaaaaattt aaattataat
     5941 tatttttata gcacgtgatg aaaaggaccc aggtggcact tttcggggaa atgtgcgcgg
     6001 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     6061 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     6121 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     6181 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     6241 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     6301 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     6361 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     6421 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     6481 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     6541 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     6601 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     6661 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     6721 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     6781 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     6841 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     6901 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     6961 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     7021 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     7081 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     7141 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     7201 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     7261 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     7321 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     7381 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     7441 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     7501 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     7561 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     7621 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     7681 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     7741 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     7801 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     7861 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     7921 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     7981 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
     8041 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     8101 tcaca
//
