LOCUS       sequence-256190-        8106 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 8106)
  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 8106)
  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
FEATURES             Location/Qualifiers
     source          1..8106
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    113..134
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(149..154,155..172,173..179)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    187..203
                     /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     192..214
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     211..227
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     211..227
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     246..266
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        248..266
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        317..666
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /label=MET17 promoter
                     /note="expression is repressed by methionine"
     primer_bind     667..683
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     667..683
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2206..2222)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2207..2223)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      2225..2472
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2241..2259)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2490..2509)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2491..2509)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2519..2536)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2519..2535)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2528..2550)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2676..3131
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2763..2782)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2973..2994
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3432..3839
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             3840..4934
                     /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(5428..5447)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5547..5569
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5607..5625)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    5667..6170
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        6207..6311
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6312..6380,6381..7172)
                     /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(6530..6549)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7343..7931
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7832..7851
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8085..8102
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
       61 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
      121 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
      181 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
      241 agcgcgcaat taaccctcac taaagggaac aaaagctgga gctccggatg caagggttcg
      301 aatcccttag ctctcattat tttttgcttt ttctcttgag gtcacatgat cgcaaaatgg
      361 caaatggcac gtgaagctgt cgatattggg gaactgtggt ggttggcaaa tgactaatta
      421 agttagtcaa ggcgccatcc tcatgaaaac tgtgtaacat aataaccgaa gtgtcgaaaa
      481 ggtggcacct tgtccaattg aacacgctcg atgaaaaaaa taagatatat ataaggttaa
      541 gtaaagcgtc tgttagaaag gaagtttttc ctttttcttg ctctcttgtc ttttcatcta
      601 ctatttcctt cgtgtaatac agggtcgtca gatacataga tacaattcta ttacccccat
      661 ccatactcta gaactagtgg atccgcgatg agtaacgatg aaggagaaac atttgccacg
      721 gaacagacta ctcaacaagt ctttcagaag ctaggtagca acatggaaaa tcgagtttgt
      781 tttgattgtg gtaacaagaa tcctacttgg acttcggtac cttttggtgt gatgctgtgt
      841 attcaatgct ctgctgtgca tagaaacatg ggtgttcata tcacctttgt aaagtcttct
      901 acattagata aatggacaat taacaattta agaaggttta agttgggcgg gaaccataag
      961 gctcgtgatt ttttcttaaa gaacaatggg aaacagctac taaatacagc aaatgttgac
     1021 gcgaaaacaa aatatacgag ccctgttgct aagaaatata agatacatct cgacaagaag
     1081 gttcaaaagg atatggagtt atacccatca gaattagtct tgaacggtca agattcttcg
     1141 gactcaccgt tggacaccga ttcagacgct tccaggtcga cttccaaaga aaatagtgtt
     1201 gacgattttt tctctaattg gcaaaaacca agctccaatt ccagttctaa actcaatgta
     1261 aatacaggct ctctcgcccc aaaaaacaac accacaggca gcacacccaa gaccactgtc
     1321 actaaaacga gaagttccat cttaacggct agcagaaaga aaccggtatt aaactcgcaa
     1381 gataaaaaga aacattcaat tttgtcttcg tcaagaaagc ccactagact aaccgcaaag
     1441 aaagtcgaca aatcacaggc agaagacttg tttgatcaat ttaaaaaaga ggctcaacaa
     1501 gaaaaggagg acgagttcac taattcgtct tctagcacca aaattagaca aaatgattac
     1561 gattcccaat ttatgaataa cagtaaaggc aacaataata acagtataga tgatattaat
     1621 acccagcctg atgaattcaa tgatttttta aatgacactt caaacagctt cgacacaact
     1681 agaaaagaac agcaagatac tttaacaccc aagtttgcca aattgggatt tggtatgacc
     1741 atgaatgacg ccaatgattt ggcaaagcaa caaaaagaat ctcagaaaat agctcaggga
     1801 ccaagatata cgggcagaat cgcggaaagg tacggcacac aaaaagctat atcatctgat
     1861 caattatttg gtcgtggctc atttgatgaa gcagcaaaca gggaagcgca tgacaagttg
     1921 aaaacatttg acaatgctac atcaatttcc tcttcttcat actttggaga agataaagaa
     1981 gtggatgaat ttggtaatcc catcaatagt agtggaagtg gagcagggaa ttttgatggc
     2041 cgtaattcta ataacggttt catcgatttt aatgcctccg cagatgatga attgcagatg
     2101 ttgagagacg ttgtagaaca gggtgctgaa aaactcggta gttatttaag ggactactta
     2161 agaaaataat ccccccgggc tgcaggaatt cgatatcaag cttatcgata ccgtcgacct
     2221 cgagtcatgt aattagttat gtcacgctta cattcacgcc ctccccccac atccgctcta
     2281 accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt ttttatagtt
     2341 atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct gtacagacgc
     2401 gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg acgctcgaag
     2461 gctttaattt gcggccggta cccaattcgc cctatagtga gtcgtattac gcgcgctcac
     2521 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
     2581 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
     2641 cttcccaaca gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag
     2701 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2761 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2821 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2881 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2941 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     3001 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     3061 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     3121 gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     3181 atatcgaccg gtcgaggaga acttctagta tatctacata cctaatatta ttgccttatt
     3241 aaaaatggaa tcccaacaat tacatcaaaa tccacattct cttcaaaatc aattgtcctg
     3301 tacttccttg ttcatgtgtg ttcaaaaacg ttatatttat aggataatta tactctattt
     3361 ctcaacaagt aattggttgt ttggccgagc ggtctaaggc gcctgattca agaaatatct
     3421 tgaccgcagt taactgtggg aatactcagg tatcgtaaga tgcaagagtt cgaatctctt
     3481 agcaaccatt atttttttcc tcaacataac gagaacacac aggggcgcta tcgcacagaa
     3541 tcaaattcga tgactggaaa ttttttgtta atttcagagg tcgcctgacg catatacctt
     3601 tttcaactga aaaattggga gaaaaaggaa aggtgagagc gccggaaccg gcttttcata
     3661 tagaatagag aagcgttcat gactaaatgc ttgcatcaca atacttgaag ttgacaatat
     3721 tatttaagga cctattgttt tttccaatag gtggttagca atcgtcttac tttctaactt
     3781 ttcttacctt ttacatttca gcaatatata tatatatatt tcaaggatat accattctaa
     3841 tgtctgcccc taagaagatc gtcgttttgc caggtgacca cgttggtcaa gaaatcacag
     3901 ccgaagccat taaggttctt aaagctattt ctgatgttcg ttccaatgtc aagttcgatt
     3961 tcgaaaatca tttaattggt ggtgctgcta tcgatgctac aggtgttcca cttccagatg
     4021 aggcgctgga agcctccaag aaggctgatg ccgttttgtt aggtgctgtg ggtggtccta
     4081 aatggggtac cggtagtgtt agacctgaac aaggtttact aaaaatccgt aaagaacttc
     4141 aattgtacgc caacttaaga ccatgtaact ttgcatccga ctctctttta gacttatctc
     4201 caatcaagcc acaatttgct aaaggtactg acttcgttgt tgtcagagaa ttagtgggag
     4261 gtatttactt tggtaagaga aaggaagacg atggtgatgg tgtcgcttgg gatagtgaac
     4321 aatacaccgt tccagaagtg caaagaatca caagaatggc cgctttcatg gccctacaac
     4381 atgagccacc attgcctatt tggtccttgg ataaagctaa tgttttggcc tcttcaagat
     4441 tatggagaaa aactgtggag gaaaccatca agaacgaatt ccctacattg aaggttcaac
     4501 atcaattgat tgattctgcc gccatgatcc tagttaagaa cccaacccac ctaaatggta
     4561 ttataatcac cagcaacatg tttggtgata tcatctccga tgaagcctcc gttatcccag
     4621 gttccttggg tttgttgcca tctgcgtcct tggcctcttt gccagacaag aacaccgcat
     4681 ttggtttgta cgaaccatgc cacggttctg ctccagattt gccaaagaat aaggtcaacc
     4741 ctatcgccac tatcttgtct gctgcaatga tgttgaaatt gtcattgaac ttgcctgaag
     4801 aaggtaaggc cattgaagat gcagttaaaa aggttttgga tgcaggtatc agaactggtg
     4861 atttaggtgg ttccaacagt accaccgaag tcggtgatgc tgtcgccgaa gaagttaaga
     4921 aaatccttgc ttaaaaagat tctctttttt tatgatattt gtacataaac tttataaatg
     4981 aaattcataa tagaaacgac acgaaattac aaaatggaat atgttcatag ggtagacgaa
     5041 actatatacg caatctacat acatttatca agaaggagaa aaaggaggat gtaaaggaat
     5101 acaggtaagc aaattgatac taatggctca acgtgataag gaaaaagaat tgcactttaa
     5161 cattaatatt gacaaggagg agggcaccac acaaaaagtt aggtgtaaca gaaaatcatg
     5221 aaactatgat tcctaattta tatattggag gattttctct aaaaaaaaaa aaatacaaca
     5281 aataaaaaac actcaatgac ctgaccattt gatggagttt aagtcaatac cttcttgaac
     5341 catttcccat aatggtgaaa gttccctcaa gaattttact ctgtcagaaa cggccttaac
     5401 gacgtagtcg atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     5461 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     5521 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     5581 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     5641 catgataata atggtttctt aggacggatc gcttgcctgt aacttacacg cgcctcgtat
     5701 cttttaatga tggaataatt tgggaattta ctctgtgttt atttattttt atgttttgta
     5761 tttggatttt agaaagtaaa taaagaaggt agaagagtta cggaatgaag aaaaaaaaat
     5821 aaacaaaggt ttaaaaaatt tcaacaaaaa gcgtacttta catatatatt tattagacaa
     5881 gaaaagcaga ttaaatagat atacattcga ttaacgataa gtaaaatgta aaatcacagg
     5941 attttcgtgt gtggtcttct acacagacaa gatgaaacaa ttcggcatta atacctgaga
     6001 gcaggaagag caagataaaa ggtagtattt gttggcgatc cccctagagt cttttacatc
     6061 ttcggaaaac aaaaactatt ttttctttaa tttctttttt tactttctat ttttaattta
     6121 tatatttata ttaaaaaatt taaattataa ttatttttat agcacgtgat gaaaaggacc
     6181 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     6241 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     6301 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     6361 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     6421 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     6481 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     6541 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     6601 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     6661 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     6721 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     6781 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     6841 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     6901 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     6961 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     7021 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     7081 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     7141 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     7201 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     7261 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     7321 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     7381 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     7441 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     7501 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     7561 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     7621 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     7681 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     7741 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     7801 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     7861 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     7921 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     7981 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     8041 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     8101 aggaag
//
