LOCUS       sequence-256192-        8106 bp    DNA     circular SYN 15-APR-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 Apr 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 Delta-2x+ve
FEATURES             Location/Qualifiers
     source          1..8106
                     /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     351..367
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     351..367
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1890..1906)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1891..1907)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      1909..2156
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1925..1943)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2174..2193)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2175..2193)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2203..2220)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2203..2219)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2212..2234)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2360..2815
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2447..2466)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2657..2678
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3116..3523
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             3524..4618
                     /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(5112..5131)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5231..5253
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5291..5309)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    5351..5854
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5891..5995
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5996..6064,6065..6856)
                     /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(6214..6233)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7027..7615
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7516..7535
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7769..7786
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7903..7924
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7939..7944,7945..7962,7963..7969)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7977..7993
                     /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     7982..8004
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8001..8017
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8001..8017
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     8036..8056
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        8038..8056
                     /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 tctagaacta
      361 gtggatccgc gatgagtaac gatgaaggag aaacatttgc cacggaacag actactcaac
      421 aagtttttca gaagctaggt agcaacatgg aaaatcgagt ttgttttgat tgtggtaaca
      481 agaatcctac ttggacttcg gtaccttttg gtgtgatgct gtgtattcaa tgctctgctg
      541 tgcataagaa catgggtgtt catatcacct ttgtaaagtc ttctacatta gataaatgga
      601 caattaacaa tttaagaagg tttaagttgg gtgggaacca taaggctcgt gattttttct
      661 taaagaacaa tgggaaacag ctactaaata cagcaaatgt tgacgcgaaa acaaaatata
      721 cgagccctgt tgctaagaaa tataagatac atctcgacaa gaaggttcaa aaggatatgg
      781 agttataccc atcagaatta gtcttgaacg gtcaagattc ttcggactca ccgttggaca
      841 ccgattcaga cgcttccagg tcgacttcca aagaaaatag tgttgacgac tttttctcta
      901 attggcaaaa accaagctcc aattccagtt ctaaactcaa tgtaaataca ggctctctcg
      961 ccccaaaaaa caacaccaca ggcagcacac ccaagaccac tgtcactaaa acgagaagtt
     1021 ccatcttaac ggctagcgaa gaggaaccgg tattaaactc gcaagatgaa gaggaacatt
     1081 caattttgtc ttcgtcaaga aagcccacta gactaaccgc aaagaaagtc gacaaatcac
     1141 aggcagaaga cttgtttgat caatttaaaa aagaggctca acaagaaaag gaggacgagt
     1201 tcactaattc gtcttctagc accaaaatta gacaaaatga ttacgattcc caatttatga
     1261 ataacagtaa aggcaacaat aataacagta tagatgatat taatacccag cctgatgaat
     1321 tcaatgattt tttaaatgac acttcaaaca gcttcgacac aactagaaaa gaacagcaag
     1381 atactttaac acccaagttt gccaaattgg gatttggtat gaccatgaat gacgccaatg
     1441 atttggcaaa gcaacaaaaa gaatctcaga aaatagctca gggaccaaga tatacgggca
     1501 gaatcgcgga aaggtacggc acacaaaaag ctatatcatc tgatcaatta tttggtcgtg
     1561 gctcatttga tgaagcagca aacagggaag cgcatgacaa gttgaaaaca tttgacaatg
     1621 ctacatcaat ttcctcttct tcatactttg gagaagataa agaagtggat gaatttggta
     1681 atcccatcaa tagtagtgga agtggagcag ggaattttga tggccgtaat tctaataacg
     1741 gtttcatcga ttttaatgcc tccgcagatg atgaattgca gatgttgaga gacgttgtag
     1801 aacagggtgc tgaaaaactc ggtagttatt taagggacta cttaagaaaa taatcccccc
     1861 gggctgcagg aattcgatat caagcttatc gataccgtcg acctcgagtc atgtaattag
     1921 ttatgtcacg cttacattca cgccctcccc ccacatccgc tctaaccgaa aaggaaggag
     1981 ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa
     2041 cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca
     2101 ttatactgaa aaccttgctt gagaaggttt tgggacgctc gaaggcttta atttgcggcc
     2161 ggtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca
     2221 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     2281 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     2341 cagcctgaat ggcgaatgga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     2401 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     2461 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     2521 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     2581 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     2641 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     2701 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     2761 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttcctga
     2821 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatcg accggtcgag
     2881 gagaacttct agtatatcta catacctaat attattgcct tattaaaaat ggaatcccaa
     2941 caattacatc aaaatccaca ttctcttcaa aatcaattgt cctgtacttc cttgttcatg
     3001 tgtgttcaaa aacgttatat ttataggata attatactct atttctcaac aagtaattgg
     3061 ttgtttggcc gagcggtcta aggcgcctga ttcaagaaat atcttgaccg cagttaactg
     3121 tgggaatact caggtatcgt aagatgcaag agttcgaatc tcttagcaac cattattttt
     3181 ttcctcaaca taacgagaac acacaggggc gctatcgcac agaatcaaat tcgatgactg
     3241 gaaatttttt gttaatttca gaggtcgcct gacgcatata cctttttcaa ctgaaaaatt
     3301 gggagaaaaa ggaaaggtga gagcgccgga accggctttt catatagaat agagaagcgt
     3361 tcatgactaa atgcttgcat cacaatactt gaagttgaca atattattta aggacctatt
     3421 gttttttcca ataggtggtt agcaatcgtc ttactttcta acttttctta ccttttacat
     3481 ttcagcaata tatatatata tatttcaagg atataccatt ctaatgtctg cccctaagaa
     3541 gatcgtcgtt ttgccaggtg accacgttgg tcaagaaatc acagccgaag ccattaaggt
     3601 tcttaaagct atttctgatg ttcgttccaa tgtcaagttc gatttcgaaa atcatttaat
     3661 tggtggtgct gctatcgatg ctacaggtgt tccacttcca gatgaggcgc tggaagcctc
     3721 caagaaggct gatgccgttt tgttaggtgc tgtgggtggt cctaaatggg gtaccggtag
     3781 tgttagacct gaacaaggtt tactaaaaat ccgtaaagaa cttcaattgt acgccaactt
     3841 aagaccatgt aactttgcat ccgactctct tttagactta tctccaatca agccacaatt
     3901 tgctaaaggt actgacttcg ttgttgtcag agaattagtg ggaggtattt actttggtaa
     3961 gagaaaggaa gacgatggtg atggtgtcgc ttgggatagt gaacaataca ccgttccaga
     4021 agtgcaaaga atcacaagaa tggccgcttt catggcccta caacatgagc caccattgcc
     4081 tatttggtcc ttggataaag ctaatgtttt ggcctcttca agattatgga gaaaaactgt
     4141 ggaggaaacc atcaagaacg aattccctac attgaaggtt caacatcaat tgattgattc
     4201 tgccgccatg atcctagtta agaacccaac ccacctaaat ggtattataa tcaccagcaa
     4261 catgtttggt gatatcatct ccgatgaagc ctccgttatc ccaggttcct tgggtttgtt
     4321 gccatctgcg tccttggcct ctttgccaga caagaacacc gcatttggtt tgtacgaacc
     4381 atgccacggt tctgctccag atttgccaaa gaataaggtc aaccctatcg ccactatctt
     4441 gtctgctgca atgatgttga aattgtcatt gaacttgcct gaagaaggta aggccattga
     4501 agatgcagtt aaaaaggttt tggatgcagg tatcagaact ggtgatttag gtggttccaa
     4561 cagtaccacc gaagtcggtg atgctgtcgc cgaagaagtt aagaaaatcc ttgcttaaaa
     4621 agattctctt tttttatgat atttgtacat aaactttata aatgaaattc ataatagaaa
     4681 cgacacgaaa ttacaaaatg gaatatgttc atagggtaga cgaaactata tacgcaatct
     4741 acatacattt atcaagaagg agaaaaagga ggatgtaaag gaatacaggt aagcaaattg
     4801 atactaatgg ctcaacgtga taaggaaaaa gaattgcact ttaacattaa tattgacaag
     4861 gaggagggca ccacacaaaa agttaggtgt aacagaaaat catgaaacta tgattcctaa
     4921 tttatatatt ggaggatttt ctctaaaaaa aaaaaaatac aacaaataaa aaacactcaa
     4981 tgacctgacc atttgatgga gtttaagtca ataccttctt gaaccatttc ccataatggt
     5041 gaaagttccc tcaagaattt tactctgtca gaaacggcct taacgacgta gtcgatatgg
     5101 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     5161 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     5221 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     5281 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     5341 tcttaggacg gatcgcttgc ctgtaactta cacgcgcctc gtatctttta atgatggaat
     5401 aatttgggaa tttactctgt gtttatttat ttttatgttt tgtatttgga ttttagaaag
     5461 taaataaaga aggtagaaga gttacggaat gaagaaaaaa aaataaacaa aggtttaaaa
     5521 aatttcaaca aaaagcgtac tttacatata tatttattag acaagaaaag cagattaaat
     5581 agatatacat tcgattaacg ataagtaaaa tgtaaaatca caggattttc gtgtgtggtc
     5641 ttctacacag acaagatgaa acaattcggc attaatacct gagagcagga agagcaagat
     5701 aaaaggtagt atttgttggc gatcccccta gagtctttta catcttcgga aaacaaaaac
     5761 tattttttct ttaatttctt tttttacttt ctatttttaa tttatatatt tatattaaaa
     5821 aatttaaatt ataattattt ttatagcacg tgatgaaaag gacccaggtg gcacttttcg
     5881 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5941 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     6001 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     6061 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     6121 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     6181 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     6241 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     6301 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     6361 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     6421 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     6481 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     6541 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     6601 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     6661 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     6721 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     6781 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     6841 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     6901 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     6961 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     7021 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     7081 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     7141 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     7201 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     7261 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     7321 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     7381 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     7441 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     7501 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     7561 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     7621 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     7681 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     7741 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     7801 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     7861 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     7921 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     7981 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat
     8041 taaccctcac taaagggaac aaaagctgga gctccggatg caagggttcg aatcccttag
     8101 ctctca
//
