LOCUS       sequence-368315-        5977 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CAG promoter-driven expression of BioID2-fused protein (N terminus).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5977)
  AUTHORS   Oura S, Ninomiya A, Sugihara F, Matzuk MM, Ikawa M
  TITLE     Proximity-dependent biotin labeling in testicular germ cells 
            identified TESMIN-associated proteins.
  JOURNAL   Sci Rep. 2022 Dec 23;12(1):22198. doi: 10.1038/s41598-022-26501-7.
  PUBMED    36564444
REFERENCE   2  (bases 1 to 5977)
  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: "Sci Rep."; 
            date: "2022-12-23"; volume: "12(1):22198. doi"; pages: " 
            10.1038/s41598-022-26501-7"
COMMENT     Sequence Label: pCAG-3xFLAG-BioID2-MCS
FEATURES             Location/Qualifiers
     source          1..5977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        17..396
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          675..1689
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1615..1637
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1697..1716
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1744..1753
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1753..1818
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1834..2529
                     /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"
     primer_bind     complement(2657..2676)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2722..2777
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2776..2795)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3132..3151)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3133..3151)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3161..3178)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3161..3177)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3170..3192)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3319..3774
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3406..3425)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3616..3637
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3800..3904
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3905..3973,3974..4765)
                     /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(4123..4142)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4936..5524
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5425..5444
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5678..5695
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5812..5833
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5848..5853,5854..5871,5872..5878)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5886..5902
                     /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     5891..5913
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5910..5926
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5910..5926
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5945..5965
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5947..5965
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 gagctcatcg atactcgaca ttgattattg actagttatt aatagtaatc aattacgggg
       61 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
      121 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
      181 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      241 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      301 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
      361 cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc
      421 ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa
      481 ttattttgtg cagcgatggg ggcggggggg gggggggncg cgcgccrggs ggggsggggs
      541 ggggsgrggg gsggggsggg gsgrggcgga gaggkgcggc ggcagccaat cagagcggcg
      601 cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa
      661 gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg
      721 cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg
      781 acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc
      841 tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct
      901 cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg
      961 gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg
     1021 ggagcgcggc cgggggcggt gccccgcggt gcgggggggg ctgcgagggg aacaaaggct
     1081 gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc
     1141 aaccccccct gcacccccct ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc
     1201 tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg caggtggggg
     1261 tgccgggcgg ggcggggccg cctcgggccg gggrgggctc gggggagggg cgcggcggcc
     1321 cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat
     1381 cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg tgcggagccg aaatctggga
     1441 ggcgccgccg caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg
     1501 aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc
     1561 ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg
     1621 gcttctggcg tgtgaccggc ggctctagcc tctgctaacc atgttcatgc cttcttcttt
     1681 ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaatct
     1741 agagccgcca tggactacaa agaccatgac ggtgattata aagatcatga cattgattac
     1801 aaggatgacg atgacaaggg gggcggggct agcttcaaga acctgatctg gctgaaggag
     1861 gtggacagca cccaggagag actgaaggag tggaacgtga gctacggcac cgccctggtg
     1921 gccgacagac agaccaaggg cagaggcggc ctgggcagaa agtggctgag ccaggagggc
     1981 ggcctgtact tcagcttcct gctgaacccc aaggagttcg agaacctgct gcagctgccc
     2041 ctggtgctgg gcctgagcgt gagcgaggcc ctggaggaga tcaccgagat ccccttcagc
     2101 ctgaagtggc ccaacgacgt gtacttccag gagaagaagg tgagcggcgt gctgtgcgag
     2161 ctgagcaagg acaagctgat cgtgggcatc ggcatcaacg tgaaccagag agagatcccc
     2221 gaggagatca aggacagagc caccaccctg tacgagatca ccggcaagga ctgggacaga
     2281 aaggaggtgc tgctgaaggt gctgaagaga atcagcgaga acctgaagaa gttcaaggag
     2341 aagagcttca aggagttcaa gggcaagatc gagagcaaga tgctgtacct gggcgaggag
     2401 gtgaagctgc tgggcgaggg caagatcacc ggcaagctgg tgggcctgag cgagaagggc
     2461 ggcgccctga tcctgaccga ggagggcatc aaggagatcc tgagcggcga gttcagcctg
     2521 agaagaagcg gatccctgca ggaattcgat atcaagcttg tcgacgcggc cgcctcgaga
     2581 ctcctcaggt gcaggctgcc tatcagaagg tggtggctgg tgtggccaat gccctggctc
     2641 acaaatacca ctgagatctt tttccctctg ccaaaaatta tggggacatc atgaagcccc
     2701 ttgagcatct gacttctggc taataaagga aatttatttt cattgcaata gtgtgttgga
     2761 attttttgtg tctctcactc ggaaggacat atgggagggc aaatcattta aaacatcaga
     2821 atgagtattt ggtttagagt ttggcaacat atgcccatat gctggctgcc atgaacaaag
     2881 gttggctata aagaggtcat cagtatatga aacagccccc tgctgtccat tccttattcc
     2941 atagaaaagc cttgacttga ggttagattt tttttatatt ttgttttgtg ttattttttt
     3001 ctttaacatc cctaaaattt tccttacatg ttttactagc cagatttttc ctcctctcct
     3061 gactactccc agtcatagct gtccctcttc tcttatgaag atccctcgac ttaattaagg
     3121 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
     3181 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     3241 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     3301 gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     3361 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     3421 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     3481 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     3541 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     3601 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     3661 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     3721 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     3781 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     3841 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     3901 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     3961 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     4021 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     4081 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     4141 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     4201 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     4261 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     4321 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     4381 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     4441 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     4501 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     4561 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     4621 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     4681 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     4741 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     4801 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     4861 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     4921 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     4981 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     5041 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     5101 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     5161 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     5221 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     5281 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     5341 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     5401 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     5461 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     5521 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     5581 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     5641 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     5701 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     5761 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     5821 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     5881 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
     5941 gcgcgcaatt aaccctcact aaagggaaca aaagctg
//
