3xHA-TurboID-NLS_pCDNA3

LOCUS        3xHA-TurboID-NLS_pCDNA3               6524 bp ds-DNA     circular SYN 20-DEC-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 Dec 20, 2021 from SnapGene Server 1.1.58
            http://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..6524
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(19..38)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     265..285
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(294..722)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(404..425)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     616..635
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    768..992
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     981..998
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     promoter        1018..1036
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1018..1035
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(1067..1087)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(1091..1111)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(1115..1135)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(1154..2110)
                     /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             complement(2117..2143)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(2147..2173)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(2177..2203)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2231..2250)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2232..2250)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2295..2498)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2324..2344)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2499..2878
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3050..3069
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3144..3248
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3249..4109
                     /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(3467..3486)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4280..4868
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4769..4788
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5022..5039
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5156..5177
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5192..5222
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5230..5246
                     /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     5235..5257
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5254..5270
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5254..5270
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    5319..5440
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5330..5349)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5384..5403
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(5614..6408)
                     /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(5725..5744)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6335..6354
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(6475..280)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6489..100
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 gctcagaggc agaggcggcc tcggcctctg cataaataaa aaaaattagt cagccatggg
       61 gcggagaatg ggcggaactg ggcggagtta ggggcgggat gggcggagtt aggggcggga
      121 ctatggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg
      181 gggactttcc acacctggtt gctgactaat tgagatgcat gctttgcata cttctgcctg
      241 ctggggagcc tggggacttt ccacacccta actgacacac attccacaga attaattcgc
      301 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
      361 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
      421 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
      481 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
      541 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
      601 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
      661 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
      721 gtggggatac cccctagagc cccagctggt tctttccgcc tcagaagcca tagagcccac
      781 cgcatcccca gcatgcctgc tattgtcttc ccaatcctcc cccttgctgt cctgccccac
      841 cccacccccc agaatagaat gacacctact cagacaatgc gatgcaattt cctcatttta
      901 ttaggaaagg acagtgggag tggcaccttc cagggtcaag gaaggcacgg gggaggggca
      961 aacaacagat ggctggcaac tagaaggcac agtcgaggct gatcagcgag ctctagcatt
     1021 taggtgacac tatagaatag ggccctctag atgcatgctc gagtcacacc ttcctcttct
     1081 tcttggggtc caccttcctc ttcttcttgg ggtccacctt cctcttcttc ttggggtcgg
     1141 ccctgctgaa ttccttttcg gcagaccgca gactgatttc tccgcccatc cagggtttga
     1201 taactccgtc ctgttccagc agcagggctc cctgtttatc aatcccccgg ctaatcccga
     1261 aaatctcttt gtccccaatg atcagcttca cgggtctatt gatgaagtta tccagcttct
     1321 cccaccgtgg cagatatgga gccaggcctt cctgctcgaa cagttccagg gctgcccgca
     1381 gctctcggat cagcgtagcg gccagagtat tcctgtccag gttaatccct gcttcctgca
     1441 gtgtgatcca gccctgattg accacgcttt cctccacgcg cctcatagcc acgttaatcc
     1501 cggcgccaat gacgatctgt gcagcatcgc ctgttattcc ggccagctcc accaggatgc
     1561 ctgccagctt tctatcctgc agatacaggt cattgggcca tttgactcgc accttgtctg
     1621 ctcccagctt tcgcagcgct tctgccatga caattccgat gaccgggccc aggccgattg
     1681 ctgctggtcc ccgcttcagg cgccaaaaca tactcaggta caggttagct ccaaaaggag
     1741 agaaccattt cctccctctg cttcctctcc ctgcctgctg atattctgca atgcaagcat
     1801 ccccactctt cagctcgccg attcgatcca gcaggtactg attggtggag tcgaccacag
     1861 gcaggactgc cacgctcccg ccgtccagct gtcccagaat ctgtttagcg ttcagcagcg
     1921 ggataggctc gggcagagag tagccctttc cgggcacggt gaacacgtcc acgccccagt
     1981 cgcgcagagt ctgaatgtgc ttgttgatag cggccctgga catgcccagg gtttctccca
     2041 gctgttcgcc actatggaac tcgccattag ccaggagagc gatcagcttc agaggcacag
     2101 tattgtcttt gctagctgcg tagtctggga cgtcataggg gtacccggca tagtcgggca
     2161 cgtcgtaggg atagccagcg taatccggaa catcatacgg gtacatggtg gcggccgcaa
     2221 gcttgggtct ccctatagtg agtcgtatta atttcgataa gccagtaagc agtgggttct
     2281 ctagttagcc agagagctct gcttatatag acctcccacc gtacacgcct accgcccatt
     2341 tgcgtcaatg gggcggagtt gttacgacat tttggaaagt cccgttgatt ttggtgccaa
     2401 aacaaactcc cattgacgtc aatggggtgg agacttggaa atccccgtga gtcaaaccgc
     2461 tatccacgcc cattgatgta ctgccaaaac cgcatcacca tggtaatagc gatgactaat
     2521 acgtagatgt actgccaagt aggaaagtcc cataaggtca tgtactgggc ataatgccag
     2581 gcgggccatt taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga
     2641 tgtactgcca agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtc
     2701 cctattggcg ttactatggg aacatacgtc attattgacg tcaatgggcg ggggtcgttg
     2761 ggcggtcagc caggcgggcc atttaccgta agttatgtaa cgcggaactc catatatggg
     2821 ctatgaacta atgaccccgt aattgattac tattaataac tagtcaataa tcaatgtcaa
     2881 cgcgtatatc tggcccgtac atcgcgaagc agcgcaaaac gcctaaccct aagcagattc
     2941 ttcatgcaat tgtcggtcaa gccttgcctt gttgtagctt aaattttgct cgcgcactac
     3001 tcagcgacct ccaacacaca agcagggagc agatactggc ttaactatgc ggcatcagag
     3061 cagattgtac tgagagtgca ccatagggga tcgggagatc tcccgatccg tcgacgtcag
     3121 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     3181 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     3241 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     3301 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     3361 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     3421 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     3481 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     3541 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     3601 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     3661 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     3721 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     3781 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     3841 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     3901 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     3961 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     4021 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     4081 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     4141 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     4201 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     4261 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     4321 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     4381 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     4441 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4501 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     4561 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     4621 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     4681 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     4741 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4801 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4861 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4921 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4981 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     5041 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     5101 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     5161 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     5221 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     5281 ccaagctcta gctagaggtc gacggtatac agacatgata agatacattg atgagtttgg
     5341 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     5401 tgctttattt gtaaccatta taagctgcaa taaacaagtt ggggtgggcg aagaactcca
     5461 gcatgagatc cccgcgctgg aggatcatcc agccggcgtc ccggaaaacg attccgaagc
     5521 ccaacctttc atagaaggcg gcggtggaat cgaaatctcg tgatggcagg ttgggcgtcg
     5581 cttggtcggt catttcgaac cccagagtcc cgctcagaag aactcgtcaa gaaggcgata
     5641 gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa agcacgagga agcggtcagc
     5701 ccattcgccg ccaagctctt cagcaatatc acgggtagcc aacgctatgt cctgatagcg
     5761 gtccgccaca cccagccggc cacagtcgat gaatccagaa aagcggccat tttccaccat
     5821 gatattcggc aagcaggcat cgccatgggt cacgacgaga tcctcgccgt cgggcatgcg
     5881 cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt cgtccagatc
     5941 atcctgatcg acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc
     6001 ttggtggtcg aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc
     6061 catgatggat actttctcgg caggagcaag gtgagatgac aggagatcct gccccggcac
     6121 ttcgcccaat agcagccagt cccttcccgc ttcagtgaca acgtcgagca cagctgcgca
     6181 aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc tcgtcctgca gttcattcag
     6241 ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa
     6301 cacggcggca tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc
     6361 cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc
     6421 tcatcctgtc tcttgatcag atccgaaaat ggatatacaa gctcccggga gctttttgca
     6481 aaagcctagg cctccaaaaa agcctcctca ctacttctgg aata
//