LOCUS       sequence-256074-        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 A
FEATURES             Location/Qualifiers
     source          1..8852
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(2285..2301)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2286..2302)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      2304..2551
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2320..2338)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2569..2588)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2570..2588)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2598..2615)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2598..2614)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2607..2629)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2755..3210
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2842..2861)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3052..3073
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3511..3918
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             3919..5013
                     /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(5507..5526)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5626..5648
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5686..5704)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    5746..6249
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        6286..6390
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6391..6459,6460..7251)
                     /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(6609..6628)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7422..8010
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7911..7930
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8164..8181
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8298..8319
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8334..8339,8340..8357,8358..8364)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8372..8388
                     /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     8377..8399
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8396..8412
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8396..8412
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     8431..8451
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        8433..8451
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        8502..8851
                     /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 gggcagaatc gcggaaaggt acggcacaca
     1921 aaaagctata gcatctgatc aattatttgg tcgtggcgca tttgatgaag cagcaaacag
     1981 ggaagcgcat gacaagttga aaacatttga caatgctaca tcaatttcct cttcttcata
     2041 ctttggagaa gataaagaag tggatgaatt tggtaatccc atcaatagta gtggaagtgg
     2101 agcagggaat tttgatggcc gtaattctaa taacggtttc atcgatttta atgcctccgc
     2161 agatgatgaa ttgcagatgt tgagagacgt tgtagaacag ggtgctgaga aactcggtag
     2221 ttatttaagg gactacttaa gaaaataatc cccccgggct gcaggaattc gatatcaagc
     2281 ttatcgatac cgtcgacctc gagtcatgta attagttatg tcacgcttac attcacgccc
     2341 tccccccaca tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc
     2401 tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct
     2461 tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa
     2521 ggttttggga cgctcgaagg ctttaatttg cggccggtac ccaattcgcc ctatagtgag
     2581 tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     2641 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     2701 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc
     2761 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     2821 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     2881 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     2941 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     3001 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     3061 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     3121 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     3181 attttaacaa aatattaacg cttacaattt cctgatgcgg tattttctcc ttacgcatct
     3241 gtgcggtatt tcacaccgca tatcgaccgg tcgaggagaa cttctagtat atctacatac
     3301 ctaatattat tgccttatta aaaatggaat cccaacaatt acatcaaaat ccacattctc
     3361 ttcaaaatca attgtcctgt acttccttgt tcatgtgtgt tcaaaaacgt tatatttata
     3421 ggataattat actctatttc tcaacaagta attggttgtt tggccgagcg gtctaaggcg
     3481 cctgattcaa gaaatatctt gaccgcagtt aactgtggga atactcaggt atcgtaagat
     3541 gcaagagttc gaatctctta gcaaccatta tttttttcct caacataacg agaacacaca
     3601 ggggcgctat cgcacagaat caaattcgat gactggaaat tttttgttaa tttcagaggt
     3661 cgcctgacgc atataccttt ttcaactgaa aaattgggag aaaaaggaaa ggtgagagcg
     3721 ccggaaccgg cttttcatat agaatagaga agcgttcatg actaaatgct tgcatcacaa
     3781 tacttgaagt tgacaatatt atttaaggac ctattgtttt ttccaatagg tggttagcaa
     3841 tcgtcttact ttctaacttt tcttaccttt tacatttcag caatatatat atatatattt
     3901 caaggatata ccattctaat gtctgcccct aagaagatcg tcgttttgcc aggtgaccac
     3961 gttggtcaag aaatcacagc cgaagccatt aaggttctta aagctatttc tgatgttcgt
     4021 tccaatgtca agttcgattt cgaaaatcat ttaattggtg gtgctgctat cgatgctaca
     4081 ggtgttccac ttccagatga ggcgctggaa gcctccaaga aggctgatgc cgttttgtta
     4141 ggtgctgtgg gtggtcctaa atggggtacc ggtagtgtta gacctgaaca aggtttacta
     4201 aaaatccgta aagaacttca attgtacgcc aacttaagac catgtaactt tgcatccgac
     4261 tctcttttag acttatctcc aatcaagcca caatttgcta aaggtactga cttcgttgtt
     4321 gtcagagaat tagtgggagg tatttacttt ggtaagagaa aggaagacga tggtgatggt
     4381 gtcgcttggg atagtgaaca atacaccgtt ccagaagtgc aaagaatcac aagaatggcc
     4441 gctttcatgg ccctacaaca tgagccacca ttgcctattt ggtccttgga taaagctaat
     4501 gttttggcct cttcaagatt atggagaaaa actgtggagg aaaccatcaa gaacgaattc
     4561 cctacattga aggttcaaca tcaattgatt gattctgccg ccatgatcct agttaagaac
     4621 ccaacccacc taaatggtat tataatcacc agcaacatgt ttggtgatat catctccgat
     4681 gaagcctccg ttatcccagg ttccttgggt ttgttgccat ctgcgtcctt ggcctctttg
     4741 ccagacaaga acaccgcatt tggtttgtac gaaccatgcc acggttctgc tccagatttg
     4801 ccaaagaata aggtcaaccc tatcgccact atcttgtctg ctgcaatgat gttgaaattg
     4861 tcattgaact tgcctgaaga aggtaaggcc attgaagatg cagttaaaaa ggttttggat
     4921 gcaggtatca gaactggtga tttaggtggt tccaacagta ccaccgaagt cggtgatgct
     4981 gtcgccgaag aagttaagaa aatccttgct taaaaagatt ctcttttttt atgatatttg
     5041 tacataaact ttataaatga aattcataat agaaacgaca cgaaattaca aaatggaata
     5101 tgttcatagg gtagacgaaa ctatatacgc aatctacata catttatcaa gaaggagaaa
     5161 aaggaggatg taaaggaata caggtaagca aattgatact aatggctcaa cgtgataagg
     5221 aaaaagaatt gcactttaac attaatattg acaaggagga gggcaccaca caaaaagtta
     5281 ggtgtaacag aaaatcatga aactatgatt cctaatttat atattggagg attttctcta
     5341 aaaaaaaaaa aatacaacaa ataaaaaaca ctcaatgacc tgaccatttg atggagttta
     5401 agtcaatacc ttcttgaacc atttcccata atggtgaaag ttccctcaag aattttactc
     5461 tgtcagaaac ggccttaacg acgtagtcga tatggtgcac tctcagtaca atctgctctg
     5521 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     5581 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     5641 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     5701 tatttttata ggttaatgtc atgataataa tggtttctta ggacggatcg cttgcctgta
     5761 acttacacgc gcctcgtatc ttttaatgat ggaataattt gggaatttac tctgtgttta
     5821 tttattttta tgttttgtat ttggatttta gaaagtaaat aaagaaggta gaagagttac
     5881 ggaatgaaga aaaaaaaata aacaaaggtt taaaaaattt caacaaaaag cgtactttac
     5941 atatatattt attagacaag aaaagcagat taaatagata tacattcgat taacgataag
     6001 taaaatgtaa aatcacagga ttttcgtgtg tggtcttcta cacagacaag atgaaacaat
     6061 tcggcattaa tacctgagag caggaagagc aagataaaag gtagtatttg ttggcgatcc
     6121 ccctagagtc ttttacatct tcggaaaaca aaaactattt tttctttaat ttcttttttt
     6181 actttctatt tttaatttat atatttatat taaaaaattt aaattataat tatttttata
     6241 gcacgtgatg aaaaggaccc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     6301 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     6361 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     6421 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     6481 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     6541 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     6601 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     6661 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     6721 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     6781 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     6841 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     6901 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     6961 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     7021 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     7081 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     7141 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     7201 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     7261 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     7321 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     7381 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     7441 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     7501 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     7561 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     7621 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     7681 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     7741 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     7801 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     7861 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     7921 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     7981 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     8041 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     8101 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     8161 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     8221 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     8281 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     8341 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     8401 aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag
     8461 ctggagctcc ggatgcaagg gttcgaatcc cttagctctc attatttttt gctttttctc
     8521 ttgaggtcac atgatcgcaa aatggcaaat ggcacgtgaa gctgtcgata ttggggaact
     8581 gtggtggttg gcaaatgact aattaagtta gtcaaggcgc catcctcatg aaaactgtgt
     8641 aacataataa ccgaagtgtc gaaaaggtgg caccttgtcc aattgaacac gctcgatgaa
     8701 aaaaataaga tatatataag gttaagtaaa gcgtctgtta gaaaggaagt ttttcctttt
     8761 tcttgctctc ttgtcttttc atctactatt tccttcgtgt aatacagggt cgtcagatac
     8821 atagatacaa ttctattacc cccatccata ct
//
