LOCUS       sequence-238509-        6112 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To fuse your protein of interest to the N-terminus of BioID2 and use
            in proximity-dependent biotin identification (BioID); MCS is present
            upstream of BioID2 tag and HA tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6112)
  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 6112)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Biol 
            Cell. 2016 Feb 24. pii: mbc.E15-12-0844."
COMMENT     Sequence Label: MCS-BioID2-HA
FEATURES             Location/Qualifiers
     source          1..6112
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(46..65)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        237..616
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        617..820
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     771..791
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     865..884
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        865..883
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             953..1648
                     /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             1649..1675
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(1705..1722)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1711..1935
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1981..2409
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2068..2087)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2278..2299
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2418..2438)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2423..2752
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2603..2738
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2665..2684
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2819..3613
                     /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(2873..2892)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3483..3502
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3787..3908
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3824..3843)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3878..3897
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3957..3973)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3957..3973)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3970..3992)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3981..3997
                     /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(join(4005..4011,4012..4029,4030..4035))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4050..4071
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4188..4205)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4359..4947)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4439..4458)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5118..5909,5910..5978))
                     /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     5741..5760
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5979..6083)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
       61 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
      121 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
      181 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      241 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      301 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      361 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      421 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      481 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      541 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      601 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      661 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      721 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      781 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc
      841 cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc aagctggcta
      901 gcgcttaagg cctgttaacc ggtcgtacgt ctccggattc gaattcggat ccttcaagaa
      961 cctgatctgg ctgaaggagg tggacagcac ccaggagaga ctgaaggagt ggaacgtgag
     1021 ctacggcacc gccctggtgg ccgacagaca gaccaagggc agaggcggcc tgggcagaaa
     1081 gtggctgagc caggagggcg gcctgtactt cagcttcctg ctgaacccca aggagttcga
     1141 gaacctgctg cagctgcccc tggtgctggg cctgagcgtg agcgaggccc tggaggagat
     1201 caccgagatc cccttcagcc tgaagtggcc caacgacgtg tacttccagg agaagaaggt
     1261 gagcggcgtg ctgtgcgagc tgagcaagga caagctgatc gtgggcatcg gcatcaacgt
     1321 gaaccagaga gagatccccg aggagatcaa ggacagagcc accaccctgt acgagatcac
     1381 cggcaaggac tgggacagaa aggaggtgct gctgaaggtg ctgaagagaa tcagcgagaa
     1441 cctgaagaag ttcaaggaga agagcttcaa ggagttcaag ggcaagatcg agagcaagat
     1501 gctgtacctg ggcgaggagg tgaagctgct gggcgagggc aagatcaccg gcaagctggt
     1561 gggcctgagc gagaagggcg gcgccctgat cctgaccgag gagggcatca aggagatcct
     1621 gagcggcgag ttcagcctga gaagaagcta cccttacgat gtaccggatt acgcatagaa
     1681 gcttaagttt aaaccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     1741 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     1801 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     1861 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     1921 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc
     1981 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     2041 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     2101 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     2161 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     2221 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     2281 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     2341 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     2401 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     2461 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     2521 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     2581 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     2641 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     2701 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     2761 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat
     2821 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     2881 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     2941 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     3001 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     3061 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     3121 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     3181 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     3241 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     3301 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     3361 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     3421 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     3481 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     3541 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     3601 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     3661 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     3721 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     3781 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3841 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     3901 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     3961 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4021 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4081 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4141 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4201 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4261 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4321 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4381 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4441 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4501 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4561 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4621 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4681 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4741 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4801 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4861 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4921 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4981 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5041 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5101 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5161 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5221 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5281 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5341 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5401 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5461 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5521 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5581 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5641 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5701 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5761 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5821 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5881 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5941 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6001 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6061 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cg
//
