LOCUS       sequence-338517-        6561 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AviTag-DogTag-Maltose binding protein in bacterial 
            cytoplasm. The AviTag enables site-specific biotinylation by BirA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6561)
  AUTHORS   Keeble AH, Yadav VK, Ferla MP, Bauer CC, Chuntharpursat-Bon E, Huang
            J, Bon RS, Howarth M
  TITLE     DogCatcher allows loop-friendly protein-protein ligation.
  JOURNAL   Cell Chem Biol. 2021 Jul 28. pii: S2451-9456(21)00315-9. doi: 
            10.1016/j.chembiol.2021.07.005.
  PUBMED    34324879
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: "Cell Chem 
            Biol. 2021 Jul 28. pii: S2451-9456(21)00315-9. doi: 
            10.1016/j.chembiol.2021.07.005."
COMMENT     Sequence Label: pET28-AviTag-DogTag-MBP
FEATURES             Location/Qualifiers
     source          1..6561
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(35..1117)
                     /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     1079..1098
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1118..1195)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1401..1420
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1504..1523
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1504..1522
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1523..1547
                     /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)."
     RBS             1562..1584
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1603..1620
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1630..1647
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1654..1698
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             1708..1776
                     /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="DIPATYEFTDGKHYITNEPIPPK"
     CDS             1795..2892
                     /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     2866..2889
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2929..2946
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2999..3017)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3013..3060
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3097..3552
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3184..3203)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3394..3415
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3644..4459)
                     /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             complement(3644..4459)
                     /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     4367..4386
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4451..4470
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4581..5169
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5070..5089
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5323..5340
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5355..5494
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5420..5439)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5580..5602
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5596..5787)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
       61 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
      121 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
      181 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
      241 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
      301 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
      361 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
      421 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
      481 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
      541 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
      601 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
      661 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
      721 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
      781 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
      841 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
      901 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
      961 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     1021 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     1081 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     1141 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     1201 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     1261 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     1321 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     1381 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     1441 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
     1501 aattaatacg actcactata ggggaattgt gagcggataa caattcccct atagaaataa
     1561 ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac
     1621 agcagcggcc tggtgccgcg cggcagccat atgggtctga atgatatttt cgaagcgcag
     1681 aaaattgaat ggcatgaagg tagcggagat attccggcta catacgaatt caccgatggt
     1741 aaacattata tcaccaatga accgataccg ccgaaaggta gtggtgaaag tggtaaaatc
     1801 gaagaaggta aactggtaat ctggattaac ggcgataaag gctataacgg tctcgctgaa
     1861 gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca tccggataaa
     1921 ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat tatcttctgg
     1981 gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat caccccggac
     2041 aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta caacggcaag
     2101 ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa agatctgctg
     2161 ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa
     2221 ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc gctgattgct
     2281 gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa agacgtgggc
     2341 gtggataacg ctggcgcgaa agcgggtctg accttcctgg ttgacctgat taaaaacaaa
     2401 cacatgaatg cagacaccga ttactccatc gcagaagctg cctttaataa aggcgaaaca
     2461 gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa agtgaattat
     2521 ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt tggcgtgctg
     2581 agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt cctcgaaaac
     2641 tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct gggtgccgta
     2701 gcgctgaagt cttacgagga agagttggcg aaagatccac gtattgccgc cactatggaa
     2761 aacgcccaga aaggtgaaat catgccgaac atcccgcaga tgtccgcttt ctggtatgcc
     2821 gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa
     2881 gacgcgcaga ctaattcgag ctcgtaaaag cttgcggccg cactcgagca ccaccaccac
     2941 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     3001 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     3061 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     3121 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3181 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3241 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3301 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     3361 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     3421 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     3481 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     3541 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     3601 tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga
     3661 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     3721 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     3781 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     3841 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     3901 gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     3961 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
     4021 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     4081 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
     4141 atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     4201 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
     4261 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
     4321 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
     4381 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt
     4441 cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta
     4501 ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     4561 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     4621 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     4681 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     4741 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     4801 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     4861 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     4921 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4981 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     5041 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     5101 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     5161 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     5221 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     5281 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     5341 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     5401 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     5461 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     5521 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     5581 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     5641 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     5701 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     5761 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     5821 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     5881 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     5941 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     6001 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     6061 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     6121 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     6181 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     6241 catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt
     6301 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag
     6361 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag
     6421 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     6481 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     6541 cggtcgagat cccggtgcct a
//
