LOCUS       sequence-404693-        6561 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of maltose binding protein with a peptide tag 
            for covalent bond formation and an AviTag for site-specific 
            biotinylation..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6561)
  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 6561)
  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 AviTag-DogTag2-MBP
FEATURES             Location/Qualifiers
     source          1..6561
                     /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(194..1291)
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     complement(197..220)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             complement(1310..1378)
                     /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"
     CDS             complement(1388..1432)
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             complement(1439..1456)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1466..1483)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1502..1524
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1539..1563
                     /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(1563..1582)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1564..1582)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1666..1685)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1891..1968
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1969..3051
                     /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
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(1988..2007)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3860..4051
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4045..4067)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4153..4292
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4208..4227
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4307..4324)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4478..5066)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4558..4577)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5177..5196)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5188..6003
                     /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             5188..6003
                     /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(5261..5280)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6095..6550)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6232..6253)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6444..6463
                     /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 acgagctcga attagtctgc gcgtctttca gggcttcatc gacagtctga cgaccgctgg
      241 cggcgttgat caccgcagta cgcacggcat accagaaagc ggacatctgc gggatgttcg
      301 gcatgatttc acctttctgg gcgttttcca tagtggcggc aatacgtgga tctttcgcca
      361 actcttcctc gtaagacttc agcgctacgg cacccagcgg tttgtcttta ttaaccgctt
      421 ccagaccttc atcagtcagc agatagtttt cgaggaactc ttttgccagc tctttgttcg
      481 gactggcggc gttaatacct gcgctcagca cgccaacgaa cggtttggat ggttgaccct
      541 tgaaggtcgg cagtaccgtt acaccataat tcactttgct ggtgtcgatg ttggaccatg
      601 cccacgggcc gttgatggtc atcgctgttt cgcctttatt aaaggcagct tctgcgatgg
      661 agtaatcggt gtctgcattc atgtgtttgt ttttaatcag gtcaaccagg aaggtcagac
      721 ccgctttcgc gccagcgtta tccacgccca cgtctttaat gtcgtacttg ccgttttcat
      781 acttgaacgc ataacccccg tcagcagcaa tcagcggcca ggtgaagtac ggttcttgca
      841 ggttgaacat cagcgcgctc ttacctttcg ctttcagttc tttatccagc gccgggatct
      901 cttcccaggt ttttggcggg ttcggcagca gatctttgtt ataaatcagc gataacgctt
      961 caacagcgat cgggtaagca atcagcttgc cgttgtaacg tacggcatcc caggtaaacg
     1021 gatacagctt gtcctggaac gctttgtccg gggtgatttc agccaacagg ccagattgag
     1081 cgtagccacc aaagcggtcg tgtgcccaga agataatgtc agggccatcg ccagttgccg
     1141 caacctgtgg gaatttctct tccagtttat ccggatgctc aacggtgact ttaattccgg
     1201 tatctttctc gaatttctta ccgacttcag cgagaccgtt atagccttta tcgccgttaa
     1261 tccagattac cagtttacct tcttcgattt taccactttc accactacct ttcggcggca
     1321 gcggttcatt ggtgatataa tgtttaccat cggtgaattc gtatgtagcc ggaatatctc
     1381 cgctaccttc atgccattca attttctgcg cttcgaaaat atcattcaga cccatatggc
     1441 tgccgcgcgg caccaggccg ctgctgtgat gatgatgatg atggctgctg cccatggtat
     1501 atctccttct taaagttaaa caaaattatt tctatagggg aattgttatc cgctcacaat
     1561 tcccctatag tgagtcgtat taatttcgcg ggatcgagat ctcgatcctc tacgccggac
     1621 gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc tggcgcctat atcgccgaca
     1681 tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg
     1741 gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg catgcaccat
     1801 tccttgcggc ggcggtgctc aacggcctca acctactact gggctgcttc ctaatgcagg
     1861 agtcgcataa gggagagcgt cgagatcccg gacaccatcg aatggcgcaa aacctttcgc
     1921 ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta
     1981 acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg
     2041 aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag
     2101 ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt
     2161 ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa
     2221 tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc
     2281 gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt
     2341 aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg
     2401 gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa
     2461 gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg
     2521 ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat
     2581 ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc
     2641 ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt
     2701 gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt
     2761 ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg
     2821 ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg
     2881 ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg
     2941 aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     3001 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     3061 aattaatgta agttagctca ctcattaggc accgggatct cgaccgatgc ccttgagagc
     3121 cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg ccgcacttat
     3181 gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct gggtcatttt
     3241 cggcgaggac cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg cggtattcgg
     3301 aatcttgcac gccctcgctc aagccttcgt cactggtccc gccaccaaac gtttcggcga
     3361 gaagcaggcc attatcgccg gcatggcggc cccacgggtg cgcatgatcg tgctcctgtc
     3421 gttgaggacc cggctaggct ggcggggttg ccttactggt tagcagaatg aatcaccgat
     3481 acgcgagcga acgtgaagcg actgctgctg caaaacgtct gcgacctgag caacaacatg
     3541 aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag cgccctgcac
     3601 cattatgttc cggatctgca tcgcaggatg ctgctggcta ccctgtggaa cacctacatc
     3661 tgtattaacg aagcgctggc attgaccctg agtgattttt ctctggtccc gccgcatcca
     3721 taccgccagt tgtttaccct cacaacgttc cagtaaccgg gcatgttcat catcagtaac
     3781 ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt acccccatga acagaaatcc
     3841 cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt
     3901 tatcagaagc cagacattaa cgcttctgga gaaactcaac gagctggacg cggatgaaca
     3961 ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca gctgcctcgc
     4021 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     4081 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     4141 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt
     4201 aactatgcgg catcagagca gattgtactg agagtgcacc attgcggtgt gaaataccgc
     4261 acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact
     4321 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     4381 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     4441 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4501 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4561 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4621 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4681 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4741 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4801 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     4861 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     4921 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     4981 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5041 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5101 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gaacaataaa actgtctgct
     5161 tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac gtcttgctct
     5221 aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat
     5281 aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag
     5341 ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga
     5401 ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct
     5461 gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca ggtattagaa
     5521 gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg
     5581 cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag
     5641 gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat
     5701 ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat
     5761 tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta
     5821 ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc
     5881 ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat
     5941 ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc
     6001 taagaattaa ttcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     6061 ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta
     6121 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     6181 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     6241 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     6301 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     6361 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     6421 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     6481 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     6541 cagggcgcgt cccattcgcc a
//
