LOCUS       Exported                7704 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 7704)
  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 7704)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7704
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             13..1107
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(1600..1619)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1719..1741
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1779..1797)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    1839..2342
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        2379..2483
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2484..3344
                     /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(2702..2721)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3515..4103
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4004..4023
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4257..4274
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4391..4412
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4427..4457
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4465..4481
                     /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     4470..4492
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4489..4505
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4489..4505
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4524..4544
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4526..4544
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        4747..5188
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    4747..4864
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     5159..5182
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             5263..6033
                     /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             6040..6081
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     terminator      6094..6341
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(6110..6128)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(6359..6378)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6360..6378)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6388..6405)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6388..6404)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6397..6419)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6545..7000
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6632..6651)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6842..6863
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7300..7704
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
ORIGIN
        1 atgtctgccc ctatgtctgc ccctaagaag atcgtcgttt tgccaggtga ccacgttggt
       61 caagaaatca cagccgaagc cattaaggtt cttaaagcta tttctgatgt tcgttccaat
      121 gtcaagttcg atttcgaaaa tcatttaatt ggtggtgctg ctatcgatgc tacaggtgtc
      181 ccacttccag atgaggcgct ggaagcctcc aagaaggttg atgccgtttt gttaggtgct
      241 gtgggtggtc ctaaatgggg taccggtagt gttagacctg aacaaggttt actaaaaatc
      301 cgtaaagaac ttcaattgta cgccaactta agaccatgta actttgcatc cgactctctt
      361 ttagacttat ctccaatcaa gccacaattt gctaaaggta ctgacttcgt tgttgtcaga
      421 gaattagtgg gaggtattta ctttggtaag agaaaggaag acgatggtga tggtgtcgct
      481 tgggatagtg aacaatacac cgttccagaa gtgcaaagaa tcacaagaat ggccgctttc
      541 atggccctac aacatgagcc accattgcct atttggtcct tggataaagc taatgttttg
      601 gcctcttcaa gattatggag aaaaactgtg gaggaaacca tcaagaacga attccctaca
      661 ttgaaggttc aacatcaatt gattgattct gccgccatga tcctagttaa gaacccaacc
      721 cacctaaatg gtattataat caccagcaac atgtttggtg atatcatctc cgatgaagcc
      781 tccgttatcc caggttcctt gggtttgttg ccatctgcgt ccttggcctc tttgccagac
      841 aagaacaccg catttggttt gtacgaacca tgccacggtt ctgctccaga tttgccaaag
      901 aataaggttg accctatcgc cactatcttg tctgctgcaa tgatgttgaa attgtcattg
      961 aacttgcctg aagaaggtaa ggccattgaa gatgcagtta aaaaggtttt ggatgcaggt
     1021 atcagaactg gtgatttagg tggttccaac agtaccaccg aagtcggtga tgctgtcgcc
     1081 gaagaagtta agaaaatcct tgcttaaaaa gattctcttt ttttatgata tttgtacata
     1141 aactttataa atgaaattca taatagaaac gacacgaaat tacaaaatgg aatatgttca
     1201 tagggtagac gaaactatat acgcaatcta catacattta tcaagaagga gaaaaaggag
     1261 gatagtaaag gaatacaggt aagcaaattg atactaatgg ctcaacgtga taaggaaaaa
     1321 gaattgcact ttaacattaa tattgacaag gaggagggca ccacacaaaa agttaggtgt
     1381 aacagaaaat catgaaacta cgattcctaa tttgatattg gaggattttc tctaaaaaaa
     1441 aaaaaataca acaaataaaa aacactcaat gacctgacca tttgatggag tttaagtcaa
     1501 taccttcttg aaccatttcc cataatggtg aaagttccct caagaatttt actctgtcag
     1561 aaacggcctt acgacgtagt cgatatggtg cactctcagt acaatctgct ctgatgccgc
     1621 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     1681 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     1741 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     1801 ataggttaat gtcatgataa taatggtttc ttaggacgga tcgcttgcct gtaacttaca
     1861 cgcgcctcgt atcttttaat gatggaataa tttgggaatt tactctgtgt ttatttattt
     1921 ttatgttttg tatttggatt ttagaaagta aataaagaag gtagaagagt tacggaatga
     1981 agaaaaaaaa ataaacaaag gtttaaaaaa tttcaacaaa aagcgtactt tacatatata
     2041 tttattagac aagaaaagca gattaaatag atatacattc gattaacgat aagtaaaatg
     2101 taaaatcaca ggattttcgt gtgtggtctt ctacacagac aagatgaaac aattcggcat
     2161 taatacctga gagcaggaag agcaagataa aaggtagtat ttgttggcga tccccctaga
     2221 gtcttttaca tcttcggaaa acaaaaacta ttttttcttt aatttctttt tttactttct
     2281 atttttaatt tatatattta tattaaaaaa tttaaattat aattattttt atagcacgtg
     2341 atgaaaagga cccaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2401 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     2461 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     2521 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     2581 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     2641 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     2701 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     2761 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     2821 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     2881 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     2941 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     3001 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     3061 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     3121 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     3181 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     3241 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     3301 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     3361 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     3421 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3481 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3541 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3601 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     3661 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3721 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     3781 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     3841 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     3901 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     3961 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4021 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4081 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4141 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4201 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     4261 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
     4321 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     4381 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
     4441 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
     4501 atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc
     4561 tccgcaaaga agtttaataa tcatattaca tggcattacc accatataca tatccatata
     4621 catatccata tctaatctta cttatatgtt gtggaaatgt aaagagcccc attatcttag
     4681 cctaaaaaaa ccttctcttt ggaactttca gtaatacgct taactgctca ttgctatatt
     4741 gaagtacgga ttagaagccg ccgagcgggt gacagccctc cgaaggaaga ctctcctccg
     4801 tgcgtcctcg tcttcaccgg tcgcgttcct gaaacgcaga tgtgcctcgc gccgcactgc
     4861 tccgaacaat aaagattcta caatactagc ttttatggtt atgaagagga aaaattggca
     4921 gtaacctggc cccacaaacc ttcaaatgaa cgaatcaaat taacaaccat aggatgataa
     4981 tgcgattagt tttttagcct tatttctggg gtaattaatc agcgaagcga tgatttttga
     5041 tctattaaca gatatataaa tgcaaaaact gcataaccac tttaactaat actttcaaca
     5101 ttttcggttt gtattacttc ttattcaaat gtaataaaag tatcaacaaa aaattgttaa
     5161 tatacctcta tactttaacg tcaaggagaa aaaaccccgg atcgaattct acttcataca
     5221 ttttcaatta agatggctcg ggatccaccg gtcgccacca tgatcccgct gctgaacgct
     5281 aaacagattc tgggacagct ggacggcggg agcgtggcag tcctgcctgt ggtcgactcc
     5341 accaatcagt acctgctgga tcgaatcggc gagctgaaga gtggggatgc ttgcattgca
     5401 gaatatcagc aggcagggag aggaagcaga gggaggaaat ggttctctcc ttttggagct
     5461 aacctgtacc tgagtatgtt ttggcgcctg aagcggggac cagcagcaat cggcctgggc
     5521 ccggtcatcg gaattgtcat ggcagaagcg ctgcgaaagc tgggagcaga caaggtgcga
     5581 gtcaaatggc ccaatgacct gtatctgcag gatagaaagc tggcaggcat cctggtggag
     5641 ctggccggaa taacaggcga tgctgcacag atcgtcattg gcgccgggat taacgtggct
     5701 atgaggcgcg tggaggaaag cgtggtcaat cagggctgga tcacactgca ggaagcaggg
     5761 attaacctgg acaggaatac tctggccgct atgctgatcc gagagctgcg ggcagccctg
     5821 gaactgttcg agcaggaagg cctggctcca tatctgtcac ggtgggagaa gctggataac
     5881 ttcatcaata gacccgtgaa gctgatcatt ggggacaaag agattttcgg gattagccgg
     5941 gggattgata aacagggagc cctgctgctg gaacaggacg gagttatcaa accctggatg
     6001 ggcggagaaa tcagtctgcg gtctgccgaa aaggctagcg gcaagcccat ccccaacccc
     6061 ctgctgggcc tggacagcac ctaatagctc gagtcatgta attagttatg tcacgcttac
     6121 attcacgccc tccccccaca tccgctctaa ccgaaaagga aggagttaga caacctgaag
     6181 tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc
     6241 aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct
     6301 tgcttgagaa ggttttggga cgctcgaagg ctttaatttg cggccggtac ccaattcgcc
     6361 ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga
     6421 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     6481 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     6541 atggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     6601 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     6661 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     6721 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     6781 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     6841 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     6901 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     6961 tttaacgcga attttaacaa aatattaacg tttacaattt cctgatgcgg tattttctcc
     7021 ttacgcatct gtgcggtatt tcacaccgca tatcgacggt cgaggagaac ttctagtata
     7081 tccacatacc taatattatt gccttattaa aaatggaatc ccaacaatta catcaaaatc
     7141 cacattctct tcaaaatcaa ttgtcctgta cttccttgtt catgtgtgtt caaaaacgtt
     7201 atatttatag gataattata ctctatttct caacaagtaa ttggttgttt ggccgagcgg
     7261 tctaaggcgc ctgattcaag aaatatcttg accgcagtta actgtgggaa tactcaggta
     7321 tcgtaagatg caagagttcg aatctcttag caaccattat ttttttcctc aacataacga
     7381 gaacacacag gggcgctatc gcacagaatc aaattcgatg actggaaatt ttttgttaat
     7441 ttcagaggtc gcctgacgca tatacctttt tcaactgaaa aattgggaga aaaaggaaag
     7501 gtgagaggcc ggaaccggct tttcatatag aatagagaag cgttcatgac taaatgcttg
     7561 catcacaata cttgaagttg acaatattat ttaaggacct attgtttttt ccaataggtg
     7621 gttagcaatc gtcttacttt ctaacttttc ttacctttta catttcagca atatatatat
     7681 atatttcaag gatataccat tcta
//
