LOCUS       Exported                6335 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  To fuse your protein of interest to the N-terminus of BioID2 and use
            in proximity-dependent biotin identification (BioID); MCS and a 
            flexible 25nm GS linker are present upstream of BioID2 and HA tags..
ACCESSION   .
VERSION     .
KEYWORDS    MCS-13X Linker-BioID2-HA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6335)
  AUTHORS   Kim DI, Jensen SC, Noble KA, Kc B, Roux KH, Motamedchaboki K, Roux 
            KJ
  TITLE     An improved smaller biotin ligase for BioID proximity labeling.
  JOURNAL   Mol Biol Cell. 2016 Feb 24. pii: mbc.E15-12-0844.
  PUBMED    26912792
REFERENCE   2  (bases 1 to 6335)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6335
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1182..1877
                     /codon_start=1
                     /product="promiscuous R40G mutant of a biotin protein 
                     ligase from Aquifex aeolicus (Kim et al., 2016)"
                     /label=BioID2
                     /translation="FKNLIWLKEVDSTQERLKEWNVSYGTALVADRQTKGRGGLGRKWL
                     SQEGGLYFSFLLNPKEFENLLQLPLVLGLSVSEALEEITEIPFSLKWPNDVYFQEKKVS
                     GVLCELSKDKLIVGIGINVNQREIPEEIKDRATTLYEITGKDWDRKEVLLKVLKRISEN
                     LKKFKEKSFKEFKGKIESKMLYLGEEVKLLGEGKITGKLVGLSEKGGALILTEEGIKEI
                     LSGEFSLRRS"
     CDS             1878..1904
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(1926..1943)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1932..2155
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2201..2629
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2288..2307)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2498..2519
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2638..2658)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2643..2973
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2824..2959
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2886..2905
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3040..3834
                     /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(3094..3113)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3704..3723
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4008..4129
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4045..4064)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4099..4118
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4178..4194)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4178..4194)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4191..4213)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4202..4218
                     /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(4226..4256)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4271..4292
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4409..4426)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4580..5168)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4660..4679)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5339..6199)
                     /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     5962..5981
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6200..6304)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gcttaaggcc tgttaaccgg tcgtacgtct ccggattcga attcggatcc catctttttt
      961 cggaattcgg atccggtgga ggcgggtctg gaggcggggg tagtggcggg ggtggaagcg
     1021 ggggtggagg cgggtcgggt ggcggaggta gcggaggcgg tggaagtgga ggcggtggaa
     1081 gtggtggagg aggttctggt ggcggaggta gcggtggcgg aggtagcgga ggcggtggaa
     1141 gtggtggcgg aggtagcgga ggcggtggat cggcgcgccg tttcaagaac ctgatctggc
     1201 tgaaggaggt ggacagcacc caggagagac tgaaggagtg gaacgtgagc tacggcaccg
     1261 ccctggtggc cgacagacag accaagggca gaggcggcct gggcagaaag tggctgagcc
     1321 aggagggcgg cctgtacttc agcttcctgc tgaaccccaa ggagttcgag aacctgctgc
     1381 agctgcccct ggtgctgggc ctgagcgtga gcgaggccct ggaggagatc accgagatcc
     1441 ccttcagcct gaagtggccc aacgacgtgt acttccagga gaagaaggtg agcggcgtgc
     1501 tgtgcgagct gagcaaggac aagctgatcg tgggcatcgg catcaacgtg aaccagagag
     1561 agatccccga ggagatcaag gacagagcca ccaccctgta cgagatcacc ggcaaggact
     1621 gggacagaaa ggaggtgctg ctgaaggtgc tgaagagaat cagcgagaac ctgaagaagt
     1681 tcaaggagaa gagcttcaag gagttcaagg gcaagatcga gagcaagatg ctgtacctgg
     1741 gcgaggaggt gaagctgctg ggcgagggca agatcaccgg caagctggtg ggcctgagcg
     1801 agaagggcgg cgccctgatc ctgaccgagg agggcatcaa ggagatcctg agcggcgagt
     1861 tcagcctgag aagaagctac ccttacgatg taccggatta cgcataggtt taaaccgctg
     1921 atcagcctcg actgtgcctt ctagtgccag ccatctgttg tttgcccctc ccccgtgcct
     1981 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     2041 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     2101 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     2161 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     2221 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2281 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2341 caagctctaa atcggggcat ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2401 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     2461 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     2521 acaacactca accctatctc ggtctattct tttgatttat aagggatttt ggggatttcg
     2581 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
     2641 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc aggcaggcag aagtatgcaa
     2701 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     2761 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     2821 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     2881 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     2941 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     3001 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     3061 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     3121 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     3181 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     3241 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     3301 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     3361 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     3421 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     3481 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     3541 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     3601 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     3661 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     3721 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     3781 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     3841 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     3901 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     3961 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     4021 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4081 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     4141 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     4201 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     4261 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     4321 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4381 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4441 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4501 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4561 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4621 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4681 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4741 cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt
     4801 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4861 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4921 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4981 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     5041 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5101 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5161 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5221 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5281 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5341 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5401 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5461 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5521 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5581 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5641 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5701 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5761 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5821 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5881 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5941 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6001 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6061 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6121 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6181 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6241 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6301 cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
