LOCUS       V5-miniTurbo-NES_pCDNA3                6245 bp ds-DNA     circular SYN 31-MAY-2022
DEFINITION  expresses V5-tagged miniTurbo in the mammalian cytosol.
ACCESSION   .
VERSION     .
KEYWORDS    V5-miniTurbo-NES_pCDNA3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6245)
  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 6245)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 31, 2022 from SnapGene Server 1.1.58
            //gt12365.cn
FEATURES             Location/Qualifiers
     source          1..6245
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      27..162
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     89..108
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             243..1037
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(297..316)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     907..926
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1211..1332
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1248..1267)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1302..1321
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1381..1397)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1381..1397)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1394..1416)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1405..1421
                     /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)."
     promoter        complement(1429..1459)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1474..1495
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1612..1629)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1783..2371)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1863..1882)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2542..3402)
                     /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     3165..3184
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3403..3507)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3582..3601)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3773..4152
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4153..4356
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4307..4327
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4401..4420
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4401..4419
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4439..4448
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             4448..4489
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             4496..5266
                     /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             5273..5299
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     promoter        complement(5336..5354)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5337..5354)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5374..5391)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5380..5604
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5650..6078
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5737..5756)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5947..5968
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6087..6107)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6092..176
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
ORIGIN
        1 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
       61 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
      121 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
      181 tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc
      241 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
      301 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
      361 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
      421 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
      481 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
      541 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
      601 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
      661 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
      721 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
      781 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
      841 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
      901 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
      961 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     1021 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
     1081 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     1141 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     1201 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     1261 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     1321 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     1381 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     1441 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     1501 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     1561 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     1621 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     1681 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     1741 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     1801 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     1861 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     1921 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     1981 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     2041 caaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     2101 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     2161 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     2221 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     2281 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     2341 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     2401 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     2461 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     2521 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     2581 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     2641 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     2701 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     2761 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     2821 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     2881 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     2941 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     3001 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     3061 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     3121 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     3181 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     3241 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     3301 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     3361 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     3421 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     3481 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
     3541 cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc
     3601 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
     3661 agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt
     3721 agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga
     3781 ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
     3841 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     3901 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
     3961 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     4021 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     4081 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     4141 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
     4201 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
     4261 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
     4321 aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact
     4381 gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc ttgcggccgc
     4441 caccatgggc aagcccatcc ccaaccccct gctgggcctg gacagcaccg ctagcatccc
     4501 gctgctgaac gctaaacaga ttctgggaca gctggacggc gggagcgtgg cagtcctgcc
     4561 tgtggtcgac tccaccaatc agtacctgct ggatcgaatc ggcgagctga agagtgggga
     4621 tgcttgcatt gcagaatatc agcaggcagg gagaggaagc agagggagga aatggttctc
     4681 tccttttgga gctaacctgt acctgagtat gttttggcgc ctgaagcggg gaccagcagc
     4741 aatcggcctg ggcccggtca tcggaattgt catggcagaa gcgctgcgaa agctgggagc
     4801 agacaaggtg cgagtcaaat ggcccaatga cctgtatctg caggatagaa agctggcagg
     4861 catcctggtg gagctggccg gaataacagg cgatgctgca cagatcgtca ttggcgccgg
     4921 gattaacgtg gctatgaggc gcgtggagga aagcgtggtc aatcagggct ggatcacact
     4981 gcaggaagca gggattaacc tggacaggaa tactctggcc gctatgctga tccgagagct
     5041 gcgggcagcc ctggaactgt tcgagcagga aggcctggct ccatatctgt cacggtggga
     5101 gaagctggat aacttcatca atagacccgt gaagctgatc attggggaca aagagatttt
     5161 cgggattagc cgggggattg ataaacaggg agccctgctg ctggaacagg acggagttat
     5221 caaaccctgg atgggcggag aaatcagtct gcggtctgcc gaaaagctgc agctgcctcc
     5281 cctggagcgc ctgaccctgg actaatagct cgagcatgca tctagagggc cctattctat
     5341 agtgtcacct aaatgctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc
     5401 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     5461 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     5521 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     5581 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg
     5641 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     5701 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     5761 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     5821 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     5881 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     5941 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     6001 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     6061 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     6121 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     6181 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     6241 agcaa
//