LOCUS       Exported                6338 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  expresses 3xHA-tagged miniTurbo in the mammalian nucleus.
ACCESSION   .
VERSION     .
KEYWORDS    3xHA-miniTurbo-NLS_pCDNA3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6338)
  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 6338)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6338
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     80..101
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(220..240)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        225..554
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      405..540
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     467..486
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             621..1415
                     /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(675..694)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1285..1304
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1589..1710
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1626..1645)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1680..1699
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1759..1775)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1759..1775)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1772..1794)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1783..1799
                     /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(1807..1837)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1852..1873
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1990..2007)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2161..2749)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2241..2260)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2920..3780)
                     /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     3543..3562
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3781..3885)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3960..3979)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4151..4530
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4531..4734
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4685..4705
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4779..4798
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4779..4797
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4826..4852
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4856..4882
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4886..4912
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4919..5689
                     /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             5708..5728
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5732..5752
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5756..5776
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(5807..5825)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5808..5825)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5845..5862)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5851..6075
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6121..211
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6208..6227)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
       61 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
      121 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
      181 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt
      241 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
      301 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
      361 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
      421 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
      481 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
      541 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga
      601 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
      661 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
      721 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
      781 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg
      841 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
      901 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
      961 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     1021 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     1081 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     1141 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     1201 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     1261 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     1321 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     1381 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac
     1441 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga
     1501 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     1561 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     1621 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     1681 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     1741 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     1801 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     1861 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     1921 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     1981 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     2041 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     2101 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     2161 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     2221 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     2281 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     2341 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     2401 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     2461 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     2521 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     2581 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     2641 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     2701 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     2761 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     2821 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     2881 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     2941 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     3001 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     3061 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     3121 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     3181 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     3241 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     3301 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     3361 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     3421 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     3481 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     3541 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     3601 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     3661 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     3721 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     3781 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     3841 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     3901 agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt
     3961 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
     4021 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
     4081 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
     4141 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
     4201 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     4261 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     4321 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     4381 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     4441 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     4501 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
     4561 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
     4621 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
     4681 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg
     4741 ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag
     4801 acccaagctt gcggccgcca ccatgtaccc gtatgatgtt ccggattacg ctggctatcc
     4861 ctacgacgtg cccgactatg ccgggtaccc ctatgacgtc ccagactacg cagctagcat
     4921 cccgctgctg aacgctaaac agattctggg acagctggac ggcgggagcg tggcagtcct
     4981 gcctgtggtc gactccacca atcagtacct gctggatcga atcggcgagc tgaagagtgg
     5041 ggatgcttgc attgcagaat atcagcaggc agggagagga agcagaggga ggaaatggtt
     5101 ctctcctttt ggagctaacc tgtacctgag tatgttttgg cgcctgaagc ggggaccagc
     5161 agcaatcggc ctgggcccgg tcatcggaat tgtcatggca gaagcgctgc gaaagctggg
     5221 agcagacaag gtgcgagtca aatggcccaa tgacctgtat ctgcaggata gaaagctggc
     5281 aggcatcctg gtggagctgg ccggaataac aggcgatgct gcacagatcg tcattggcgc
     5341 cgggattaac gtggctatga ggcgcgtgga ggaaagcgtg gtcaatcagg gctggatcac
     5401 actgcaggaa gcagggatta acctggacag gaatactctg gccgctatgc tgatccgaga
     5461 gctgcgggca gccctggaac tgttcgagca ggaaggcctg gctccatatc tgtcacggtg
     5521 ggagaagctg gataacttca tcaatagacc cgtgaagctg atcattgggg acaaagagat
     5581 tttcgggatt agccggggga ttgataaaca gggagccctg ctgctggaac aggacggagt
     5641 tatcaaaccc tggatgggcg gagaaatcag tctgcggtct gccgaaaagg aattcagcag
     5701 ggccgacccc aagaagaaga ggaaggtgga ccccaagaag aagaggaagg tggaccccaa
     5761 gaagaagagg aaggtgtgac tcgagcatgc atctagaggg ccctattcta tagtgtcacc
     5821 taaatgctag agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     5881 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     5941 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     6001 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     6061 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc
     6121 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     6181 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     6241 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     6301 gtgctttacg gcacctcgac cccaaaaaac ttgattag
//
