LOCUS       sequence-256187-        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 Delta-2x+ve
FEATURES             Location/Qualifiers
     source          1..8852
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     220..239
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     473..490
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    607..628
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(643..648,649..666,667..673)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    681..697
                     /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     686..708
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     705..721
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     705..721
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     740..760
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        742..760
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        811..1160
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /label=MET17 promoter
                     /note="expression is repressed by methionine"
     primer_bind     complement(3446..3462)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3447..3463)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      3465..3712
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3481..3499)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3730..3749)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3731..3749)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3759..3776)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3759..3775)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3768..3790)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3916..4371
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4003..4022)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4213..4234
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4672..5079
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             5080..6174
                     /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(6668..6687)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6787..6809
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6847..6865)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    6907..7410
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        7447..7551
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7552..7620,7621..8412)
                     /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(7770..7789)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(8583..8852,1..319)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
       61 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
      121 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
      181 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
      241 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
      301 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
      361 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
      421 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
      481 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
      541 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
      601 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
      661 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
      721 ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctccg
      781 gatgcaaggg ttcgaatccc ttagctctca ttattttttg ctttttctct tgaggtcaca
      841 tgatcgcaaa atggcaaatg gcacgtgaag ctgtcgatat tggggaactg tggtggttgg
      901 caaatgacta attaagttag tcaaggcgcc atcctcatga aaactgtgta acataataac
      961 cgaagtgtcg aaaaggtggc accttgtcca attgaacacg ctcgatgaaa aaaataagat
     1021 atatataagg ttaagtaaag cgtctgttag aaaggaagtt tttccttttt cttgctctct
     1081 tgtcttttca tctactattt ccttcgtgta atacagggtc gtcagataca tagatacaat
     1141 tctattaccc ccatccatac tctagagcat gtccaagggt gaagagctat ttactggggt
     1201 tgtacccatt ttggtagaac tggacggaga tgtaaacgga cataaattct ctgttagagg
     1261 tgagggcgaa ggcgatgcca ccaatggtaa attgactctg aagtttatat gcactacggg
     1321 taaattacct gttccttggc caaccctagt aacaactttg acatatggtg ttcaatgttt
     1381 ctcaagatac ccagaccata tgaaaaggca tgatttcttt aaaagtgcta tgccagaagg
     1441 ctacgtgcaa gagagaacta tctcctttaa ggatgacggt acgtataaaa cacgagcaga
     1501 agtgaaattc gaaggggata cactagttaa tcgcatcgaa ttaaagggta tagactttaa
     1561 ggaagatggt aatattctcg gccataaact tgagtataat ttcaactcgc ataatgtgta
     1621 cattacagct gacaaacaaa agaacggaat taaagcgaat tttaaaatca ggcacaacgt
     1681 cgaagatggg tctgttcaac ttgccgatca ttatcagcaa aacaccccta ttggtgatgg
     1741 tccagtcttg ttacctgata atcactactt aagcacacag tcgagattat ccaaagatcc
     1801 aaacgaaaag agagaccaca tggtcttgtt agaatttgtt actgctgctg gtattaccca
     1861 tggtatggat gaattgtaca aaggcggcgg cagcgccgcc gccggcggcg gcagccgcgg
     1921 atccgcgatg agtaacgatg aaggagaaac atttgccacg gaacagacta ctcaacaagt
     1981 ctttcagaag ctaggtagca acatggaaaa tcgagtttgt tttgattgtg gtaacaagaa
     2041 tcctacttgg acttcggtac cttttggtgt gatgctgtgt attcaatgct ctgctgtgca
     2101 tagaaacatg ggtgttcata tcacctttgt aaagtcttct acattagata aatggacaat
     2161 taacaattta agaaggttta agttgggcgg gaaccataag gctcgtgatt ttttcttaaa
     2221 gaacaatggg aaacagctac taaatacagc aaatgttgac gcgaaaacaa aatatacgag
     2281 ccctgttgct aagaaatata agatacatct cgacaagaag gttcaaaagg atatggagtt
     2341 atacccatca gaattagtct tgaacggtca agattcttcg gactcaccgt tggacaccga
     2401 ttcagacgct tccaggtcga cttccaaaga aaatagtgtt gacgattttt tctctaattg
     2461 gcaaaaacca agctccaatt ccagttctaa actcaatgta aatacaggct ctctcgcccc
     2521 aaaaaacaac accacaggca gcacacccaa gaccactgtc actaaaacga gaagttctat
     2581 cttaacggct agcgaagagg aaccggtatt aaactcgcaa gatgaagagg aacattcaat
     2641 tttgtcttcg tcaagaaagc ccactagact aaccgcaaag aaagtcgaca aatcacaggc
     2701 agaagacttg tttgatcaat ttaaaaaaga ggctcaacaa gaaaaggagg acgagttcac
     2761 taattcgtct tctagcacca aaattagaca aaatgattac gattcccaat ttatgaataa
     2821 cagtaaaggc aacaataata acagtataga tgatattaat acccagcctg atgaattcaa
     2881 tgatttttta aatgacactt caaacagctt cgacacaact agaaaagaac agcaagatac
     2941 tttaacaccc aagtttgcca aattgggatt tggtatgacc atgaatgacg ccaatgattt
     3001 ggcaaagcaa caaaaagaat ctcagaaaat agctcaggga ccaagatata cgggcagaat
     3061 cgcggaaagg tacggcacac aaaaagctat atcatctgat caattatttg gtcgtggctc
     3121 atttgatgaa gcagcaaaca gggaagcgca tgacaagttg aaaacatttg acaatgctac
     3181 atcaatttcc tcttcttcat actttggaga agataaagaa gtggatgaat ttggtaatcc
     3241 catcaatagt agtggaagtg gagcagggaa ttttgatggc cgtaattcta ataacggttt
     3301 catcgatttt aatgcctccg cagatgatga attgcagatg ttgagagacg ttgtagaaca
     3361 gggtgctgaa aaactcggta gttatttaag ggactactta agaaaataat ccccccgggc
     3421 tgcaggaatt cgatatcaag cttatcgata ccgtcgacct cgagtcatgt aattagttat
     3481 gtcacgctta cattcacgcc ctccccccac atccgctcta accgaaaagg aaggagttag
     3541 acaacctgaa gtctaggtcc ctatttattt ttttatagtt atgttagtat taagaacgtt
     3601 atttatattt caaatttttc ttttttttct gtacagacgc gtgtacgcat gtaacattat
     3661 actgaaaacc ttgcttgaga aggttttggg acgctcgaag gctttaattt gcggccggta
     3721 cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt
     3781 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     3841 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     3901 ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3961 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     4021 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     4081 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     4141 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     4201 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     4261 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     4321 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt tcctgatgcg
     4381 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatcgaccg gtcgaggaga
     4441 acttctagta tatctacata cctaatatta ttgccttatt aaaaatggaa tcccaacaat
     4501 tacatcaaaa tccacattct cttcaaaatc aattgtcctg tacttccttg ttcatgtgtg
     4561 ttcaaaaacg ttatatttat aggataatta tactctattt ctcaacaagt aattggttgt
     4621 ttggccgagc ggtctaaggc gcctgattca agaaatatct tgaccgcagt taactgtggg
     4681 aatactcagg tatcgtaaga tgcaagagtt cgaatctctt agcaaccatt atttttttcc
     4741 tcaacataac gagaacacac aggggcgcta tcgcacagaa tcaaattcga tgactggaaa
     4801 ttttttgtta atttcagagg tcgcctgacg catatacctt tttcaactga aaaattggga
     4861 gaaaaaggaa aggtgagagc gccggaaccg gcttttcata tagaatagag aagcgttcat
     4921 gactaaatgc ttgcatcaca atacttgaag ttgacaatat tatttaagga cctattgttt
     4981 tttccaatag gtggttagca atcgtcttac tttctaactt ttcttacctt ttacatttca
     5041 gcaatatata tatatatatt tcaaggatat accattctaa tgtctgcccc taagaagatc
     5101 gtcgttttgc caggtgacca cgttggtcaa gaaatcacag ccgaagccat taaggttctt
     5161 aaagctattt ctgatgttcg ttccaatgtc aagttcgatt tcgaaaatca tttaattggt
     5221 ggtgctgcta tcgatgctac aggtgttcca cttccagatg aggcgctgga agcctccaag
     5281 aaggctgatg ccgttttgtt aggtgctgtg ggtggtccta aatggggtac cggtagtgtt
     5341 agacctgaac aaggtttact aaaaatccgt aaagaacttc aattgtacgc caacttaaga
     5401 ccatgtaact ttgcatccga ctctctttta gacttatctc caatcaagcc acaatttgct
     5461 aaaggtactg acttcgttgt tgtcagagaa ttagtgggag gtatttactt tggtaagaga
     5521 aaggaagacg atggtgatgg tgtcgcttgg gatagtgaac aatacaccgt tccagaagtg
     5581 caaagaatca caagaatggc cgctttcatg gccctacaac atgagccacc attgcctatt
     5641 tggtccttgg ataaagctaa tgttttggcc tcttcaagat tatggagaaa aactgtggag
     5701 gaaaccatca agaacgaatt ccctacattg aaggttcaac atcaattgat tgattctgcc
     5761 gccatgatcc tagttaagaa cccaacccac ctaaatggta ttataatcac cagcaacatg
     5821 tttggtgata tcatctccga tgaagcctcc gttatcccag gttccttggg tttgttgcca
     5881 tctgcgtcct tggcctcttt gccagacaag aacaccgcat ttggtttgta cgaaccatgc
     5941 cacggttctg ctccagattt gccaaagaat aaggtcaacc ctatcgccac tatcttgtct
     6001 gctgcaatga tgttgaaatt gtcattgaac ttgcctgaag aaggtaaggc cattgaagat
     6061 gcagttaaaa aggttttgga tgcaggtatc agaactggtg atttaggtgg ttccaacagt
     6121 accaccgaag tcggtgatgc tgtcgccgaa gaagttaaga aaatccttgc ttaaaaagat
     6181 tctctttttt tatgatattt gtacataaac tttataaatg aaattcataa tagaaacgac
     6241 acgaaattac aaaatggaat atgttcatag ggtagacgaa actatatacg caatctacat
     6301 acatttatca agaaggagaa aaaggaggat gtaaaggaat acaggtaagc aaattgatac
     6361 taatggctca acgtgataag gaaaaagaat tgcactttaa cattaatatt gacaaggagg
     6421 agggcaccac acaaaaagtt aggtgtaaca gaaaatcatg aaactatgat tcctaattta
     6481 tatattggag gattttctct aaaaaaaaaa aaatacaaca aataaaaaac actcaatgac
     6541 ctgaccattt gatggagttt aagtcaatac cttcttgaac catttcccat aatggtgaaa
     6601 gttccctcaa gaattttact ctgtcagaaa cggccttaac gacgtagtcg atatggtgca
     6661 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     6721 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     6781 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     6841 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     6901 aggacggatc gcttgcctgt aacttacacg cgcctcgtat cttttaatga tggaataatt
     6961 tgggaattta ctctgtgttt atttattttt atgttttgta tttggatttt agaaagtaaa
     7021 taaagaaggt agaagagtta cggaatgaag aaaaaaaaat aaacaaaggt ttaaaaaatt
     7081 tcaacaaaaa gcgtacttta catatatatt tattagacaa gaaaagcaga ttaaatagat
     7141 atacattcga ttaacgataa gtaaaatgta aaatcacagg attttcgtgt gtggtcttct
     7201 acacagacaa gatgaaacaa ttcggcatta atacctgaga gcaggaagag caagataaaa
     7261 ggtagtattt gttggcgatc cccctagagt cttttacatc ttcggaaaac aaaaactatt
     7321 ttttctttaa tttctttttt tactttctat ttttaattta tatatttata ttaaaaaatt
     7381 taaattataa ttatttttat agcacgtgat gaaaaggacc caggtggcac ttttcgggga
     7441 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     7501 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     7561 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     7621 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     7681 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     7741 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     7801 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     7861 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     7921 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     7981 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     8041 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     8101 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     8161 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     8221 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     8281 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     8341 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     8401 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     8461 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     8521 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     8581 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     8641 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     8701 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     8761 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     8821 gctgccagtg gcgataagtc gtgtcttacc gg
//
