LOCUS       sequence-368318-        6244 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mouse Clgn promoter-driven expression of TESMIN-TurboID-3xFLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6244)
  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 6244)
  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-TurboID-3xFLAG
FEATURES             Location/Qualifiers
     source          1..6244
                     /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..2756
                     /codon_start=1
                     /product="mutant of the E. coli biotin ligase BioID 
                     engineered for improved catalytic efficiency (Branon et 
                     al., 2018)"
                     /label=TurboID
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLG
                     ADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWI
                     TLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDK
                     EIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK"
     CDS             2772..2837
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2924..2943)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2989..3044
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3043..3062)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3399..3418)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3400..3418)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3428..3445)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3428..3444)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3437..3459)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3586..4041
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3673..3692)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3883..3904
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4067..4171
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4172..4240,4241..5032)
                     /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"
     rep_origin      5203..5791
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5692..5711
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5945..5962
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6079..6100
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6115..6120,6121..6138,6139..6145)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6153..6169
                     /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     6158..6180
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6177..6193
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6177..6193
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6212..6232
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6214..6232
                     /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 gcggccgcga
     1801 aagacaatac tgtgcctctg aagctgatcg ctctcctggc taatggcgag ttccatagtg
     1861 gcgaacagct gggagaaacc ctgggcatgt ccagggccgc tatcaacaag cacattcaga
     1921 ctctgcgcga ctggggcgtg gacgtgttca ccgtgcccgg aaagggctac tctctgcccg
     1981 agcctatccc gctgctgaac gctaaacaga ttctgggaca gctggacggc gggagcgtgg
     2041 cagtcctgcc tgtggtcgac tccaccaatc agtacctgct ggatcgaatc ggcgagctga
     2101 agagtgggga tgcttgcatt gcagaatatc agcaggcagg gagaggaagc agagggagga
     2161 aatggttctc tccttttgga gctaacctgt acctgagtat gttttggcgc ctgaagcggg
     2221 gaccagcagc aatcggcctg ggcccggtca tcggaattgt catggcagaa gcgctgcgaa
     2281 agctgggagc agacaaggtg cgagtcaaat ggcccaatga cctgtatctg caggatagaa
     2341 agctggcagg catcctggtg gagctggccg gaataacagg cgatgctgca cagatcgtca
     2401 ttggcgccgg gattaacgtg gctatgaggc gcgtggagga aagcgtggtc aatcagggct
     2461 ggatcacact gcaggaagca gggattaacc tggacaggaa tactctggcc gctacgctga
     2521 tccgagagct gcgggcagcc ctggaactgt tcgagcagga aggcctggct ccatatctgc
     2581 cacggtggga gaagctggat aacttcatca atagacccgt gaagctgatc attggggaca
     2641 aagagatttt cgggattagc cgggggattg ataaacaggg agccctgctg ctggaacagg
     2701 acggagttat caaaccctgg atgggcggag aaatcagtct gcggtctgcc gaaaaggcta
     2761 gcgggggcgg ggactacaaa gaccatgacg gtgattataa agatcatgac attgattaca
     2821 aggatgacga tgacaagtag ctcgagactc ctcaggtgca ggctgcctat cagaaggtgg
     2881 tggctggtgt ggccaatgcc ctggctcaca aataccactg agatcttttt ccctctgcca
     2941 aaaattatgg ggacatcatg aagccccttg agcatctgac ttctggctaa taaaggaaat
     3001 ttattttcat tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga aggacatatg
     3061 ggagggcaaa tcatttaaaa catcagaatg agtatttggt ttagagtttg gcaacatatg
     3121 cccatatgct ggctgccatg aacaaaggtt ggctataaag aggtcatcag tatatgaaac
     3181 agccccctgc tgtccattcc ttattccata gaaaagcctt gacttgaggt tagatttttt
     3241 ttatattttg ttttgtgtta tttttttctt taacatccct aaaattttcc ttacatgttt
     3301 tactagccag atttttcctc ctctcctgac tactcccagt catagctgtc cctcttctct
     3361 tatgaagatc cctcgactta attaaggtac ccaattcgcc ctatagtgag tcgtattacg
     3421 cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
     3481 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
     3541 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg
     3601 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     3661 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     3721 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     3781 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     3841 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     3901 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     3961 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     4021 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4081 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     4141 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     4201 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     4261 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     4321 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     4381 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     4441 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     4501 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     4561 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     4621 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     4681 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     4741 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     4801 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     4861 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     4921 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     4981 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     5041 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     5101 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     5161 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     5221 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     5281 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     5341 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     5401 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     5461 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     5521 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     5581 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     5641 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     5701 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5761 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5821 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     5881 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     5941 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     6001 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     6061 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
     6121 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
     6181 aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa gggaacaaaa
     6241 gctg
//
