LOCUS       sequence-368314-        5972 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CAG promoter-driven expression of BioID2-fused protein (C terminus).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5972)
  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 5972)
  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-MCS-BioID2-3xFLAG
FEATURES             Location/Qualifiers
     source          1..5972
                     /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"
     CDS             1789..2484
                     /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             2500..2565
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2652..2671)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2717..2772
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2771..2790)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3127..3146)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3128..3146)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3156..3173)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3156..3172)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3165..3187)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3314..3769
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3401..3420)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3611..3632
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3795..3899
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3900..3968,3969..4760)
                     /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(4118..4137)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4931..5519
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5420..5439
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5673..5690
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5807..5828
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5843..5848,5849..5866,5867..5873)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5881..5897
                     /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     5886..5908
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5905..5921
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5905..5921
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5940..5960
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5942..5960
                     /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 ggggggsggg gsgrggcggr 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 gggagggctc 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 agaggatccc tgcaggaatt cgatatcaag cttgtcgacg cggccgcgtt caagaacctg
     1801 atctggctga aggaggtgga cagcacccag gagagactga aggagtggaa cgtgagctac
     1861 ggcaccgccc tggtggccga cagacagacc aagggcagag gcggcctggg cagaaagtgg
     1921 ctgagccagg agggcggcct gtacttcagc ttcctgctga accccaagga gttcgagaac
     1981 ctgctgcagc tgcccctggt gctgggcctg agcgtgagcg aggccctgga ggagatcacc
     2041 gagatcccct tcagcctgaa gtggcccaac gacgtgtact tccaggagaa gaaggtgagc
     2101 ggcgtgctgt gcgagctgag caaggacaag ctgatcgtgg gcatcggcat caacgtgaac
     2161 cagagagaga tccccgagga gatcaaggac agagccacca ccctgtacga gatcaccggc
     2221 aaggactggg acagaaagga ggtgctgctg aaggtgctga agagaatcag cgagaacctg
     2281 aagaagttca aggagaagag cttcaaggag ttcaagggca agatcgagag caagatgctg
     2341 tacctgggcg aggaggtgaa gctgctgggc gagggcaaga tcaccggcaa gctggtgggc
     2401 ctgagcgaga agggcggcgc cctgatcctg accgaggagg gcatcaagga gatcctgagc
     2461 ggcgagttca gcctgagaag aagcgctagc gggggcgggg actacaaaga ccatgacggt
     2521 gattataaag atcatgacat tgattacaag gatgacgatg acaagtagct cgagactcct
     2581 caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa
     2641 taccactgag atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag
     2701 catctgactt ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt
     2761 ttgtgtctct cactcggaag gacatatggg agggcaaatc atttaaaaca tcagaatgag
     2821 tatttggttt agagtttggc aacatatgcc catatgctgg ctgccatgaa caaaggttgg
     2881 ctataaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga
     2941 aaagccttga cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta
     3001 acatccctaa aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta
     3061 ctcccagtca tagctgtccc tcttctctta tgaagatccc tcgacttaat taaggtaccc
     3121 aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt
     3181 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     3241 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     3301 aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3361 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3421 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3481 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3541 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3601 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3661 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3721 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     3781 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     3841 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     3901 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     3961 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     4021 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     4081 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     4141 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     4201 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     4261 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     4321 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     4381 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     4441 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     4501 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     4561 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     4621 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     4681 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     4741 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     4801 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     4861 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     4921 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     4981 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     5041 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     5101 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     5161 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     5221 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     5281 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     5341 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     5401 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     5461 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     5521 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     5581 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     5641 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     5701 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     5761 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     5821 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     5881 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg
     5941 caattaaccc tcactaaagg gaacaaaagc tg
//
