LOCUS       sequence-404734-        6770 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of a modified Transglutaminase 2 with a peptide
            tag for modular ligation directed by SnoopLigase2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6770)
  AUTHORS   Keeble AH, Wood DP, Howarth M
  TITLE     Design and Evolution of Enhanced Peptide-Peptide Ligation for 
            Modular Transglutaminase Assembly.
  JOURNAL   Bioconjug Chem. 2023 Jun 8. doi: 10.1021/acs.bioconjchem.3c00122.
  PUBMED    37289810
REFERENCE   2  (bases 1 to 6770)
  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: "Bioconjug 
            Chem. 2023 Jun 8. doi: 10.1021/acs.bioconjchem.3c00122."
COMMENT     Sequence Label: pET28a-TG2x-DogTag2
FEATURES             Location/Qualifiers
     source          1..6770
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(185..214)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             complement(233..301)
                     /codon_start=1
                     /product="peptide derived from the RrgA adhesin of 
                     Streptococcus pneumoniae"
                     /label=DogTag
                     /note="forms a covalent bond with the DogCatcher protein 
                     when inserted into an intra-domain protein loop (Keeble et 
                     al., 2022)"
                     /translation="DIPATYEFTDGKHYITNEPLPPK"
     RBS             1712..1734
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1749..1773
                     /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     complement(1773..1792)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1774..1792)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1876..1895)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2101..2178
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2179..3261
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQIALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(2198..2217)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4070..4261
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4255..4277)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4363..4502
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4418..4437
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4517..4534)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4688..5275)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4768..4787)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5386..5405)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5397..6212
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             5397..6212
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5470..5489)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6304..6759)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6441..6462)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6653..6672
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagctttt
      181 attaatggtg atggtgatgg tgatggtgat ggtgaccact ttcaccacta cctttcggcg
      241 gcagcggttc attggtgata taatgtttac catcggtgaa ttcgtatgta gccggaatat
      301 cagagctgcc taatttattc aaatggtttg cccgggtgaa agcttcacgc tcttccgaag
      361 agccctcagg gtatttgtac gtgtgggtga tatcttcgcg ctcatcgcgg ccaaccgact
      421 ttgtactaat cttaaggcca acgattaacg aacgattgat cgatttatgt acagacccgt
      481 catcctgttg aatccagtca accacgtcag cgttcacctc cgcgaagaca aatggtgcat
      541 cgtactttgt ggagaggtcg ccctctttga tcgcgcggac aggcacaggg ccgcaacaat
      601 acgtcccttc acttttttcc tgaggtgttg gatccagtgc ttgccagcct tcgtaacccg
      661 gctgtaaatc cggacgtgtc atccagctct ccacccaaca atggaaattc caaatcattt
      721 cgcttttatc gccctggatc tccccaaatt cgttgcggaa atactcgatc agtaagttag
      781 agttctgatc atgtgccgag ttgtaatttg taacgacccg cgttgggata cccaaacacc
      841 gtaatacagt gcaagctact gcggcgaaca cccagcattg gccgtacttc acccgctgac
      901 aaccgtggtt cttccaacgc cgcaagatgt ccacgctacc aatccaactc atcggggaca
      961 ccccatcgcc ataattgttg tcccaccggc ccaagagaac gccctgatca tcgttgctgt
     1021 ttaccatccc tgacacaacc cgccctacgt atactggact ggaccgccgg gaacaatccc
     1081 gcccagcgtt ctttaaaaac tttgggttga cgtcaaggag aatgagacag atgtccagga
     1141 tgccgtcctc gaattgcccg aagttccacg gaatattctt aatgaacttc gcggacccct
     1201 gatagatgaa accctgttgg gtcaagacat actcttggcg ctcctcctca ctatccaaat
     1261 aaactgcatc ggccgggcac caagcgttaa acaggaggat aaaatgcccg agtacgaagc
     1321 tcgaaccttg gtaaccggtc gacgcctcca agcttaaacg gtacaaaccg atcggggcat
     1381 ttgctggagt ggtcaactgc agactaagag tgcaatcttg ttgatcgacc acggttgcag
     1441 tccaatcacc ttcctcgaca gcgtcacgca atgggaaacg ggccttcgtg cctgcttcct
     1501 gggatggggc cggaccagtt acaacactga aagttaagga gtcaacacta gcctcgtaat
     1561 tacgcccttc aaaatgtaag gttaaccaga atggttgacc acgacggacg accagctttt
     1621 cccggcagag gtcggcagta tggtggtccc gcccattcgt ctcgagttcc aagtcacagc
     1681 gctcaagaac cagttcttcg gccatggtat atctccttct taaagttaaa caaaattatt
     1741 tctagagggg aattgttatc cgctcacaat tcccctatag tgagtcgtat taatttcgcg
     1801 ggatcgagat ctcgatcctc tacgccggac gcatcgtggc cggcatcacc ggcgccacag
     1861 gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg gctcgccact
     1921 tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg gccgggggac
     1981 tgttgggcgc catctccttg catgcaccat tccttgcggc ggcggtgctc aacggcctca
     2041 acctactact gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgagatcccg
     2101 gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt
     2161 caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt
     2221 gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg
     2281 cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa
     2341 caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac
     2401 gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg
     2461 gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt
     2521 ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt
     2581 gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca
     2641 cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg
     2701 gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg
     2761 cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg
     2821 gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat
     2881 gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatagcgct gggcgcaatg
     2941 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac
     3001 gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc
     3061 ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag
     3121 ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg
     3181 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     3241 cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc
     3301 accgggatct cgaccgatgc ccttgagagc cttcaaccca gtcagctcct tccggtgggc
     3361 gcggggcatg actatcgtcg ccgcacttat gactgtcttc tttatcatgc aactcgtagg
     3421 acaggtgccg gcagcgctct gggtcatttt cggcgaggac cgctttcgct ggagcgcgac
     3481 gatgatcggc ctgtcgcttg cggtattcgg aatcttgcac gccctcgctc aagccttcgt
     3541 cactggtccc gccaccaaac gtttcggcga gaagcaggcc attatcgccg gcatggcggc
     3601 cccacgggtg cgcatgatcg tgctcctgtc gttgaggacc cggctaggct ggcggggttg
     3661 ccttactggt tagcagaatg aatcaccgat acgcgagcga acgtgaagcg actgctgctg
     3721 caaaacgtct gcgacctgag caacaacatg aatggtcttc ggtttccgtg tttcgtaaag
     3781 tctggaaacg cggaagtcag cgccctgcac cattatgttc cggatctgca tcgcaggatg
     3841 ctgctggcta ccctgtggaa cacctacatc tgtattaacg aagcgctggc attgaccctg
     3901 agtgattttt ctctggtccc gccgcatcca taccgccagt tgtttaccct cacaacgttc
     3961 cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg agcatcctct ctcgtttcat
     4021 cggtatcatt acccccatga acagaaatcc cccttacacg gaggcatcag tgaccaaaca
     4081 ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc cagacattaa cgcttctgga
     4141 gaaactcaac gagctggacg cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca
     4201 cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg
     4261 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     4321 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc
     4381 acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg
     4441 agagtgcacc attgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     4501 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     4561 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4621 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4681 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     4741 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4801 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4861 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4921 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4981 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5041 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5101 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     5161 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     5221 gcggtggttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5281 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5341 tttggtcatg aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga
     5401 gccatattca acgggaaacg tcttgctcta ggccgcgatt aaattccaac atggatgctg
     5461 atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc
     5521 gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg
     5581 ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc
     5641 cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc
     5701 ccgggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg
     5761 atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta
     5821 acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg
     5881 atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa
     5941 tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg
     6001 ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa
     6061 tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt
     6121 cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc
     6181 agtttcattt gatgctcgat gagtttttct aagaattaat tcatgagcgg atacatattt
     6241 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6301 cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     6361 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     6421 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     6481 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     6541 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     6601 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     6661 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     6721 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
//
