LOCUS       sequence-368322-        7379 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CAG promoter-driven expression of TESMIN-BioID2-3xFLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7379)
  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 7379)
  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-Tesmin-BioID2-3xFLAG
FEATURES             Location/Qualifiers
     source          1..7379
                     /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      1750..1759
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3196..3891
                     /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             3907..3972
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(4059..4078)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4124..4179
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4178..4197)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4534..4553)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4535..4553)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4563..4580)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4563..4579)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4572..4594)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4721..5176
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4808..4827)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5018..5039
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5202..5306
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5307..5375,5376..6167)
                     /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(5525..5544)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6338..6926
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6827..6846
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7080..7097
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7214..7235
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7250..7255,7256..7273,7274..7280)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7288..7304
                     /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     7293..7315
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7312..7328
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7312..7328
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7347..7367
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7349..7367
                     /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 gsgrggcggr gaggtgcggc 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 agaggatccg ccgccatgga ggacgcgctg ctcggcgcca tgactggccc cgaagacgag
     1801 ctgggcgccg agctgttcgg gtcagagcgt gtgttcgcgg acgggctggc gctgagcccc
     1861 gcaggcggtg cggccgatcg ggatgagttg ccggttctgg ccgacgccta cctgggcgcc
     1921 accgagccag gggagccgct gctgcgtgcg ctcagccccc cgccgggcgc tgaggtaccg
     1981 gccgccctgc tcggcgactt cccggggctg cccgagctcc gcagcccaga cgacgccgct
     2041 ccgcctcccg cctacagcgt gcacgtgtta tcgtcgttac ttcccggtgc tcgcgggccc
     2101 gcgctgttgc cgctaagcgc aggagtgcgc gtgatcccag ttgaaatcaa agaagcaggt
     2161 ggtagtgtgc caggcggcag ccctgaagac gcagctttcc aggcccctct ggctcaggaa
     2221 tcctgttgca agttcccatc atcccaggag gcagaggagg cctccagctg ccctcggaag
     2281 aaagactcca gccccatggt gatttgtcag ctgaaaggag gcgcccagat gctctgcata
     2341 gacaactgtg gcgcgaggga gctcaaagcg ctccatctgc ttcctcagta cgatgaccag
     2401 agcagtttcc ctcagtcaga gctccctaag ccaatgacaa ctttagtggg aagacttctg
     2461 ccagtaccag cgaagttaaa tctcatcaca caggttgata atggagctct cccatcagct
     2521 gtcaatgggg ctgcctttcc ctctggacct gctctgcaag ggccacccaa aataactctg
     2581 tctgggtact gtgactgctt ctccagcggg gacttctgca acagctgcag ctgcaacaac
     2641 ctgcgccatg agctcgagcg cttcaaagcc ataaaggcgt gtcttgatag aaatcctgaa
     2701 gctttccaac caaaaatggg gaaaggccgt ctgggagctg ctaaacttcg acacagcaaa
     2761 gggtgcaact gtaagcgctc aggctgcctg aagaactact gtgagtgcta tgaggccaaa
     2821 atcatgtgtt cttccatttg caaatgcatt gcttgcaaaa actatgaaga aagtccagaa
     2881 cgaaaaatgc tgatgagcac accccactac atggagcctg gggactttga gagcagccat
     2941 tatttgtccc cagccaagtt ctcaggacct ccaaaactga gaaaaaatag gcaggccttc
     3001 tcctgtatct cctgggaagt agtggaggcc acatgtgcct gcctgctggc ccagggtgag
     3061 gaagcagagc aggagcactg ttccccaagc ttggctgagc agatgatcct ggaggagttt
     3121 ggaaggtgcc tgtcgcagat tctccacatc gagttcaagt ccaaggggct gaaaattgag
     3181 gtcgacgcgg ccgcgttcaa gaacctgatc tggctgaagg aggtggacag cacccaggag
     3241 agactgaagg agtggaacgt gagctacggc accgccctgg tggccgacag acagaccaag
     3301 ggcagaggcg gcctgggcag aaagtggctg agccaggagg gcggcctgta cttcagcttc
     3361 ctgctgaacc ccaaggagtt cgagaacctg ctgcagctgc ccctggtgct gggcctgagc
     3421 gtgagcgagg ccctggagga gatcaccgag atccccttca gcctgaagtg gcccaacgac
     3481 gtgtacttcc aggagaagaa ggtgagcggc gtgctgtgcg agctgagcaa ggacaagctg
     3541 atcgtgggca tcggcatcaa cgtgaaccag agagagatcc ccgaggagat caaggacaga
     3601 gccaccaccc tgtacgagat caccggcaag gactgggaca gaaaggaggt gctgctgaag
     3661 gtgctgaaga gaatcagcga gaacctgaag aagttcaagg agaagagctt caaggagttc
     3721 aagggcaaga tcgagagcaa gatgctgtac ctgggcgagg aggtgaagct gctgggcgag
     3781 ggcaagatca ccggcaagct ggtgggcctg agcgagaagg gcggcgccct gatcctgacc
     3841 gaggagggca tcaaggagat cctgagcggc gagttcagcc tgagaagaag cgctagcggg
     3901 ggcggggact acaaagacca tgacggtgat tataaagatc atgacattga ttacaaggat
     3961 gacgatgaca agtagctcga gactcctcag gtgcaggctg cctatcagaa ggtggtggct
     4021 ggtgtggcca atgccctggc tcacaaatac cactgagatc tttttccctc tgccaaaaat
     4081 tatggggaca tcatgaagcc ccttgagcat ctgacttctg gctaataaag gaaatttatt
     4141 ttcattgcaa tagtgtgttg gaattttttg tgtctctcac tcggaaggac atatgggagg
     4201 gcaaatcatt taaaacatca gaatgagtat ttggtttaga gtttggcaac atatgcccat
     4261 atgctggctg ccatgaacaa aggttggcta taaagaggtc atcagtatat gaaacagccc
     4321 cctgctgtcc attccttatt ccatagaaaa gccttgactt gaggttagat tttttttata
     4381 ttttgttttg tgttattttt ttctttaaca tccctaaaat tttccttaca tgttttacta
     4441 gccagatttt tcctcctctc ctgactactc ccagtcatag ctgtccctct tctcttatga
     4501 agatccctcg acttaattaa ggtacccaat tcgccctata gtgagtcgta ttacgcgcgc
     4561 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     4621 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     4681 cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca
     4741 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     4801 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     4861 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     4921 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     4981 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     5041 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     5101 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     5161 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     5221 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     5281 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     5341 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     5401 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     5461 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     5521 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     5581 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     5641 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     5701 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     5761 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     5821 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     5881 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     5941 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     6001 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     6061 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     6121 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     6181 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6241 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6301 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6361 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6421 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6481 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     6541 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     6601 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     6661 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     6721 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     6781 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     6841 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     6901 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     6961 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     7021 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     7081 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     7141 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     7201 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
     7261 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     7321 gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctg
//
