LOCUS       Exported                6524 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  expresses 3xHA-tagged TurboID in the mammalian nucleus.
ACCESSION   .
VERSION     .
KEYWORDS    3xHA-TurboID-NLS_pCDNA3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6524)
  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 6524)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6524
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        129..508
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        509..712
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     663..683
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     757..776
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        757..775
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             804..830
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             834..860
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             864..890
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             897..1853
                     /codon_start=1
                     /product="mutant of the E. coli biotin ligase BioID 
                     engineered for improved catalytic efficiency (Branon et 
                     al., 2018)"
                     /label=TurboID
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLG
                     ADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWI
                     TLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDK
                     EIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK"
     CDS             1872..1892
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1896..1916
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1920..1940
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(1971..1989)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1972..1989)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2009..2026)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2015..2239
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2285..2713
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2372..2391)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2582..2603
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2722..2742)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2727..3056
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2907..3042
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2969..2988
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3123..3917
                     /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(3177..3196)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3787..3806
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4091..4212
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4128..4147)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4182..4201
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4261..4277)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4261..4277)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4274..4296)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4285..4301
                     /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(4309..4339)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4354..4375
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4492..4509)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4663..5251)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4743..4762)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5422..6282)
                     /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     6045..6064
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6283..6387)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6462..6481)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
       61 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
      121 tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
      181 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
      241 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
      301 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
      361 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
      421 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
      481 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
      541 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
      601 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
      661 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct
      721 aactagagaa cccactgctt actggcttat cgaaattaat acgactcact atagggagac
      781 ccaagcttgc ggccgccacc atgtacccgt atgatgttcc ggattacgct ggctatccct
      841 acgacgtgcc cgactatgcc gggtacccct atgacgtccc agactacgca gctagcaaag
      901 acaatactgt gcctctgaag ctgatcgctc tcctggctaa tggcgagttc catagtggcg
      961 aacagctggg agaaaccctg ggcatgtcca gggccgctat caacaagcac attcagactc
     1021 tgcgcgactg gggcgtggac gtgttcaccg tgcccggaaa gggctactct ctgcccgagc
     1081 ctatcccgct gctgaacgct aaacagattc tgggacagct ggacggcggg agcgtggcag
     1141 tcctgcctgt ggtcgactcc accaatcagt acctgctgga tcgaatcggc gagctgaaga
     1201 gtggggatgc ttgcattgca gaatatcagc aggcagggag aggaagcaga gggaggaaat
     1261 ggttctctcc ttttggagct aacctgtacc tgagtatgtt ttggcgcctg aagcggggac
     1321 cagcagcaat cggcctgggc ccggtcatcg gaattgtcat ggcagaagcg ctgcgaaagc
     1381 tgggagcaga caaggtgcga gtcaaatggc ccaatgacct gtatctgcag gatagaaagc
     1441 tggcaggcat cctggtggag ctggccggaa taacaggcga tgctgcacag atcgtcattg
     1501 gcgccgggat taacgtggct atgaggcgcg tggaggaaag cgtggtcaat cagggctgga
     1561 tcacactgca ggaagcaggg attaacctgg acaggaatac tctggccgct acgctgatcc
     1621 gagagctgcg ggcagccctg gaactgttcg agcaggaagg cctggctcca tatctgccac
     1681 ggtgggagaa gctggataac ttcatcaata gacccgtgaa gctgatcatt ggggacaaag
     1741 agattttcgg gattagccgg gggattgata aacagggagc cctgctgctg gaacaggacg
     1801 gagttatcaa accctggatg ggcggagaaa tcagtctgcg gtctgccgaa aaggaattca
     1861 gcagggccga ccccaagaag aagaggaagg tggaccccaa gaagaagagg aaggtggacc
     1921 ccaagaagaa gaggaaggtg tgactcgagc atgcatctag agggccctat tctatagtgt
     1981 cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     2041 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     2101 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     2161 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     2221 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat
     2281 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2341 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2401 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2461 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     2521 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     2581 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     2641 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     2701 tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     2761 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     2821 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     2881 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     2941 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
     3001 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     3061 tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc
     3121 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     3181 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     3241 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     3301 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     3361 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     3421 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     3481 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     3541 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     3601 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     3661 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     3721 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     3781 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     3841 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     3901 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
     3961 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     4021 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     4081 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     4141 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     4201 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     4261 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4321 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4381 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4441 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4501 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4561 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4621 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4681 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4741 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4801 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4861 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4921 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4981 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5041 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5101 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5161 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5221 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5281 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5341 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5401 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5461 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5521 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5581 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5641 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5701 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5761 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5821 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5881 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5941 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6001 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6061 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6121 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6181 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6241 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6301 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6361 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
     6421 cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc
     6481 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggt
//
