LOCUS       sequence-368320-        5983 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mouse Clgn promoter-driven expression of TESMIN-BioID2-3xFLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5983)
  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 5983)
  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: pClgn-Tesmin-BioID2-3xFLAG
FEATURES             Location/Qualifiers
     source          1..5983
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      354..363
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1800..2495
                     /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             2511..2576
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2663..2682)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2728..2783
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2782..2801)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3138..3157)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3139..3157)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3167..3184)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3167..3183)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3176..3198)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3325..3780
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3412..3431)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3622..3643
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3806..3910
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3911..3979,3980..4771)
                     /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(4129..4148)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4942..5530
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5431..5450
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5684..5701
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5818..5839
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5854..5859,5860..5877,5878..5884)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5892..5908
                     /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     5897..5919
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5916..5932
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5916..5932
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5951..5971
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5953..5971
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 gagctcatcg atgagctcca gagagggact gacgttggaa ggaccgcatc cccccaggcg
       61 cgcggccgct gggtcccgag cggcggttga gcgggccgct tgcgcactgg ggctgccact
      121 gcatcctgca tcctccggcg cggccgctcg cgcacgcgca gttagcagtc gctgctgggc
      181 ggctgccggc gggatcgctc tggggagacg ccgggcacca agtagcggcc agtcggacac
      241 ctgagctgcc gctgctggac acaagcccgc aggacaggtg tgtagaccgc gggccgcctt
      301 cctgcggctt gttctctggg gaccgggtga gaaatcggat ctctagagga tccgccgcca
      361 tggaggacgc gctgctcggc gccatgactg gccccgaaga cgagctgggc gccgagctgt
      421 tcgggtcaga gcgtgtgttc gcggacgggc tggcgctgag ccccgcaggc ggtgcggccg
      481 atcgggatga gttgccggtt ctggccgacg cctacctggg cgccaccgag ccaggggagc
      541 cgctgctgcg tgcgctcagc cccccgccgg gcgctgaggt accggccgcc ctgctcggcg
      601 acttcccggg gctgcccgag ctccgcagcc cagacgacgc cgctccgcct cccgcctaca
      661 gcgtgcacgt gttatcgtcg ttacttcccg gtgctcgcgg gcccgcgctg ttgccgctaa
      721 gcgcaggagt gcgcgtgatc ccagttgaaa tcaaagaagc aggtggtagt gtgccaggcg
      781 gcagccctga agacgcagct ttccaggccc ctctggctca ggaatcctgt tgcaagttcc
      841 catcatccca ggaggcagag gaggcctcca gctgccctcg gaagaaagac tccagcccca
      901 tggtgatttg tcagctgaaa ggaggcgccc agatgctctg catagacaac tgtggcgcga
      961 gggagctcaa agcgctccat ctgcttcctc agtacgatga ccagagcagt ttccctcagt
     1021 cagagctccc taagccaatg acaactttag tgggaagact tctgccagta ccagcgaagt
     1081 taaatctcat cacacaggtt gataatggag ctctcccatc agctgtcaat ggggctgcct
     1141 ttccctctgg acctgctctg caagggccac ccaaaataac tctgtctggg tactgtgact
     1201 gcttctccag cggggacttc tgcaacagct gcagctgcaa caacctgcgc catgagctcg
     1261 agcgcttcaa agccataaag gcgtgtcttg atagaaatcc tgaagctttc caaccaaaaa
     1321 tggggaaagg ccgtctggga gctgctaaac ttcgacacag caaagggtgc aactgtaagc
     1381 gctcaggctg cctgaagaac tactgtgagt gctatgaggc caaaatcatg tgttcttcca
     1441 tttgcaaatg cattgcttgc aaaaactatg aagaaagtcc agaacgaaaa atgctgatga
     1501 gcacacccca ctacatggag cctggggact ttgagagcag ccattatttg tccccagcca
     1561 agttctcagg acctccaaaa ctgagaaaaa ataggcaggc cttctcctgt atctcctggg
     1621 aagtagtgga ggccacatgt gcctgcctgc tggcccaggg tgaggaagca gagcaggagc
     1681 actgttcccc aagcttggct gagcagatga tcctggagga gtttggaagg tgcctgtcgc
     1741 agattctcca catcgagttc aagtccaagg ggctgaaaat tgaggtcgac gcggccgcgt
     1801 tcaagaacct gatctggctg aaggaggtgg acagcaccca ggagagactg aaggagtgga
     1861 acgtgagcta cggcaccgcc ctggtggccg acagacagac caagggcaga ggcggcctgg
     1921 gcagaaagtg gctgagccag gagggcggcc tgtacttcag cttcctgctg aaccccaagg
     1981 agttcgagaa cctgctgcag ctgcccctgg tgctgggcct gagcgtgagc gaggccctgg
     2041 aggagatcac cgagatcccc ttcagcctga agtggcccaa cgacgtgtac ttccaggaga
     2101 agaaggtgag cggcgtgctg tgcgagctga gcaaggacaa gctgatcgtg ggcatcggca
     2161 tcaacgtgaa ccagagagag atccccgagg agatcaagga cagagccacc accctgtacg
     2221 agatcaccgg caaggactgg gacagaaagg aggtgctgct gaaggtgctg aagagaatca
     2281 gcgagaacct gaagaagttc aaggagaaga gcttcaagga gttcaagggc aagatcgaga
     2341 gcaagatgct gtacctgggc gaggaggtga agctgctggg cgagggcaag atcaccggca
     2401 agctggtggg cctgagcgag aagggcggcg ccctgatcct gaccgaggag ggcatcaagg
     2461 agatcctgag cggcgagttc agcctgagaa gaagcgctag cgggggcggg gactacaaag
     2521 accatgacgg tgattataaa gatcatgaca ttgattacaa ggatgacgat gacaagtagc
     2581 tcgagactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc
     2641 tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga
     2701 agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt
     2761 gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac
     2821 atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga
     2881 acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct
     2941 tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat
     3001 ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc
     3061 tctcctgact actcccagtc atagctgtcc ctcttctctt atgaagatcc ctcgacttaa
     3121 ttaaggtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt
     3181 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     3241 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     3301 tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     3361 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     3421 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     3481 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     3541 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     3601 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     3661 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     3721 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     3781 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3841 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     3901 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     3961 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     4021 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     4081 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     4141 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     4201 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     4261 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     4321 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     4381 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     4441 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     4501 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     4561 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     4621 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     4681 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     4741 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     4801 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4861 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4921 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4981 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     5041 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     5101 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     5161 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     5221 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     5281 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     5341 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     5401 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     5461 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     5521 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     5581 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     5641 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     5701 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     5761 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     5821 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     5881 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     5941 cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctg
//
