LOCUS       Exported                7685 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  expresses V5-tagged miniTurbo in the yeast cytosol.
ACCESSION   .
VERSION     .
KEYWORDS    miniTurbo-V5_pRS415
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7685)
  AUTHORS   Branon TC, Bosch JA, Sanchez AD, Udeshi ND, Svinkina T, Carr SA, 
            Feldman JL, Perrimon N, Ting AY
  TITLE     Efficient proximity labeling in living cells and organisms with 
            TurboID.
  JOURNAL   Nat Biotechnol. 2018 Aug 20. pii: nbt.4201. doi: 10.1038/nbt.4201.
  PUBMED    30125270
REFERENCE   2  (bases 1 to 7685)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7685
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     23..40
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    157..178
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        193..223
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    231..247
                     /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     236..258
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     255..271
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     255..271
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     290..310
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        292..310
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        501..942
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    501..618
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     913..936
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             1017..1787
                     /codon_start=1
                     /product="N-terminally truncated mutant of the E. coli 
                     biotin ligase BioID engineered for improved catalytic 
                     efficiency (Branon et al., 2018)"
                     /label=miniTurbo
                     /translation="IPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACI
                     AEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGADK
                     VRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQ
                     EAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIF
                     GISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK"
     CDS             1794..1835
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     terminator      1848..2095
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1864..1882)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2113..2132)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2114..2132)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2142..2159)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2142..2158)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2151..2173)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2299..2754
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2386..2405)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2596..2617
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3055..3462
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             3463..4557
                     /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(5051..5070)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5170..5192
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5230..5248)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    5290..5793
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5830..5934
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5935..6795
                     /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(6153..6172)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6966..7554
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7455..7474
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
       61 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      121 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      181 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      241 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
      301 tcactaaagg gaacaaaagc tggagctccg caaagaagtt taataatcat attacatggc
      361 attaccacca tatacatatc catatctaat cttacttata tgttgtggaa atgtaaagag
      421 ccccattatc ttagcctaaa aaaaccttct ctttggaact ttcagtaata cgcttaactg
      481 ctcattgcta tattgaagta cggattagaa gccgccgagc gggtgacagc cctccgaagg
      541 aagactctcc tccgtgcgtc ctcgtcttca ccggtcgcgt tcctgaaacg cagatgtgcc
      601 tcgcgccgca ctgctccgaa caataaagat tctacaatac tagcttttat ggttatgaag
      661 aggaaaaatt ggcagtaacc tggccccaca aaccttcaaa tgaacgaatc aaattaacaa
      721 ccataggatg ataatgcgat tagtttttta gccttatttc tggggtaatt aatcagcgaa
      781 gcgatgattt ttgatctatt aacagatata taaatgcaaa aactgcataa ccactttaac
      841 taatactttc aacattttcg gtttgtatta cttcttattc aaatgtaata aaagtatcaa
      901 caaaaaattg ttaatatacc tctatacttt aacgtcaagg agaaaaaacc ccggatcgaa
      961 ttctacttca tacattttca attaagatgg ctcgggatcc accggtcgcc accatgatcc
     1021 cgctgctgaa cgctaaacag attctgggac agctggacgg cgggagcgtg gcagtcctgc
     1081 ctgtggtcga ctccaccaat cagtacctgc tggatcgaat cggcgagctg aagagtgggg
     1141 atgcttgcat tgcagaatat cagcaggcag ggagaggaag cagagggagg aaatggttct
     1201 ctccttttgg agctaacctg tacctgagta tgttttggcg cctgaagcgg ggaccagcag
     1261 caatcggcct gggcccggtc atcggaattg tcatggcaga agcgctgcga aagctgggag
     1321 cagacaaggt gcgagtcaaa tggcccaatg acctgtatct gcaggataga aagctggcag
     1381 gcatcctggt ggagctggcc ggaataacag gcgatgctgc acagatcgtc attggcgccg
     1441 ggattaacgt ggctatgagg cgcgtggagg aaagcgtggt caatcagggc tggatcacac
     1501 tgcaggaagc agggattaac ctggacagga atactctggc cgctatgctg atccgagagc
     1561 tgcgggcagc cctggaactg ttcgagcagg aaggcctggc tccatatctg tcacggtggg
     1621 agaagctgga taacttcatc aatagacccg tgaagctgat cattggggac aaagagattt
     1681 tcgggattag ccgggggatt gataaacagg gagccctgct gctggaacag gacggagtta
     1741 tcaaaccctg gatgggcgga gaaatcagtc tgcggtctgc cgaaaaggct agcggcaagc
     1801 ccatccccaa ccccctgctg ggcctggaca gcacctaata gctcgagtca tgtaattagt
     1861 tatgtcacgc ttacattcac gccctccccc cacatccgct ctaaccgaaa aggaaggagt
     1921 tagacaacct gaagtctagg tccctattta tttttttata gttatgttag tattaagaac
     1981 gttatttata tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg catgtaacat
     2041 tatactgaaa accttgcttg agaaggtttt gggacgctcg aaggctttaa tttgcggccg
     2101 gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa
     2161 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     2221 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     2281 agcctgaatg gcgaatggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     2341 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     2401 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     2461 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     2521 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     2581 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     2641 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     2701 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttcctgat
     2761 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatcga ccggtcgagg
     2821 agaacttcta gtatatctac atacctaata ttattgcctt attaaaaatg gaatcccaac
     2881 aattacatca aaatccacat tctcttcaaa atcaattgtc ctgtacttcc ttgttcatgt
     2941 gtgttcaaaa acgttatatt tataggataa ttatactcta tttctcaaca agtaattggt
     3001 tgtttggccg agcggtctaa ggcgcctgat tcaagaaata tcttgaccgc agttaactgt
     3061 gggaatactc aggtatcgta agatgcaaga gttcgaatct cttagcaacc attatttttt
     3121 tcctcaacat aacgagaaca cacaggggcg ctatcgcaca gaatcaaatt cgatgactgg
     3181 aaattttttg ttaatttcag aggtcgcctg acgcatatac ctttttcaac tgaaaaattg
     3241 ggagaaaaag gaaaggtgag agcgccggaa ccggcttttc atatagaata gagaagcgtt
     3301 catgactaaa tgcttgcatc acaatacttg aagttgacaa tattatttaa ggacctattg
     3361 ttttttccaa taggtggtta gcaatcgtct tactttctaa cttttcttac cttttacatt
     3421 tcagcaatat atatatatat atttcaagga tataccattc taatgtctgc ccctaagaag
     3481 atcgtcgttt tgccaggtga ccacgttggt caagaaatca cagccgaagc cattaaggtt
     3541 cttaaagcta tttctgatgt tcgttccaat gtcaagttcg atttcgaaaa tcatttaatt
     3601 ggtggtgctg ctatcgatgc tacaggtgtt ccacttccag atgaggcgct ggaagcctcc
     3661 aagaaggctg atgccgtttt gttaggtgct gtgggtggtc ctaaatgggg taccggtagt
     3721 gttagacctg aacaaggttt actaaaaatc cgtaaagaac ttcaattgta cgccaactta
     3781 agaccatgta actttgcatc cgactctctt ttagacttat ctccaatcaa gccacaattt
     3841 gctaaaggta ctgacttcgt tgttgtcaga gaattagtgg gaggtattta ctttggtaag
     3901 agaaaggaag acgatggtga tggtgtcgct tgggatagtg aacaatacac cgttccagaa
     3961 gtgcaaagaa tcacaagaat ggccgctttc atggccctac aacatgagcc accattgcct
     4021 atttggtcct tggataaagc taatgttttg gcctcttcaa gattatggag aaaaactgtg
     4081 gaggaaacca tcaagaacga attccctaca ttgaaggttc aacatcaatt gattgattct
     4141 gccgccatga tcctagttaa gaacccaacc cacctaaatg gtattataat caccagcaac
     4201 atgtttggtg atatcatctc cgatgaagcc tccgttatcc caggttcctt gggtttgttg
     4261 ccatctgcgt ccttggcctc tttgccagac aagaacaccg catttggttt gtacgaacca
     4321 tgccacggtt ctgctccaga tttgccaaag aataaggtca accctatcgc cactatcttg
     4381 tctgctgcaa tgatgttgaa attgtcattg aacttgcctg aagaaggtaa ggccattgaa
     4441 gatgcagtta aaaaggtttt ggatgcaggt atcagaactg gtgatttagg tggttccaac
     4501 agtaccaccg aagtcggtga tgctgtcgcc gaagaagtta agaaaatcct tgcttaaaaa
     4561 gattctcttt ttttatgata tttgtacata aactttataa atgaaattca taatagaaac
     4621 gacacgaaat tacaaaatgg aatatgttca tagggtagac gaaactatat acgcaatcta
     4681 catacattta tcaagaagga gaaaaaggag gatgtaaagg aatacaggta agcaaattga
     4741 tactaatggc tcaacgtgat aaggaaaaag aattgcactt taacattaat attgacaagg
     4801 aggagggcac cacacaaaaa gttaggtgta acagaaaatc atgaaactat gattcctaat
     4861 ttatatattg gaggattttc tctaaaaaaa aaaaaataca acaaataaaa aacactcaat
     4921 gacctgacca tttgatggag tttaagtcaa taccttcttg aaccatttcc cataatggtg
     4981 aaagttccct caagaatttt actctgtcag aaacggcctt aacgacgtag tcgatatggt
     5041 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     5101 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     5161 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     5221 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     5281 cttaggacgg atcgcttgcc tgtaacttac acgcgcctcg tatcttttaa tgatggaata
     5341 atttgggaat ttactctgtg tttatttatt tttatgtttt gtatttggat tttagaaagt
     5401 aaataaagaa ggtagaagag ttacggaatg aagaaaaaaa aataaacaaa ggtttaaaaa
     5461 atttcaacaa aaagcgtact ttacatatat atttattaga caagaaaagc agattaaata
     5521 gatatacatt cgattaacga taagtaaaat gtaaaatcac aggattttcg tgtgtggtct
     5581 tctacacaga caagatgaaa caattcggca ttaatacctg agagcaggaa gagcaagata
     5641 aaaggtagta tttgttggcg atccccctag agtcttttac atcttcggaa aacaaaaact
     5701 attttttctt taatttcttt ttttactttc tatttttaat ttatatattt atattaaaaa
     5761 atttaaatta taattatttt tatagcacgt gatgaaaagg acccaggtgg cacttttcgg
     5821 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5881 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5941 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     6001 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     6061 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     6121 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     6181 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     6241 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     6301 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     6361 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     6421 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6481 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6541 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6601 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6661 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6721 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6781 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6841 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6901 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6961 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     7021 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     7081 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     7141 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     7201 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     7261 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     7321 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     7381 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     7441 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     7501 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     7561 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     7621 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     7681 gctcg
//
