LOCUS       sequence-338524-        6279 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses in bacterial cytoplasm HaloTag7 with DogTag inserted 
            between D139 and E140, as well as C61S and C261S in HaloTag7 to 
            block disulfide bond formation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6279)
  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 6279)
  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: pET28a-HaloTag7SS-DogTag
FEATURES             Location/Qualifiers
     source          1..6279
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(778..1860)
                     /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     1822..1841
                     /label=LacI-R
                     /note="LacI, reverse primer"
     primer_bind     2145..2164
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2248..2267
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2248..2266
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2267..2291
                     /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             2306..2328
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2347..2364
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2803..2871
                     /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"
     primer_bind     complement(3461..3479)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     rep_origin      3558..4013
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3645..3664)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3855..3876
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4105..4920)
                     /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(4105..4920)
                     /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     4828..4847
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4912..4931
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5042..5630
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5531..5550
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5784..5801
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5816..5955
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5881..5900)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6041..6063
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6057..6248)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
       61 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
      121 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
      181 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
      241 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
      301 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
      361 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
      421 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
      481 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
      541 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
      601 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
      661 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
      721 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
      781 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
      841 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
      901 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
      961 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     1021 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     1081 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     1141 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     1201 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     1261 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     1321 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     1381 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     1441 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     1501 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     1561 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     1621 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     1681 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     1741 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     1801 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     1861 attcgccacc tctgacttga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     1921 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     1981 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     2041 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     2101 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     2161 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     2221 aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
     2281 ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggc
     2341 agcagccatc atcatcatca tcacagcagc ggcgaaatcg gtactggctt tccattcgac
     2401 ccccattatg tggaagtcct gggcgagcgc atgcactacg tcgatgttgg tccgcgcgat
     2461 ggcacccctg tgctgttcct gcacggtaac ccgacctcct cctacgtgtg gcgcaacatc
     2521 atcccgcatg ttgcaccgac ccatcgcagc attgctccag acctgatcgg tatgggcaaa
     2581 tccgacaaac cagacctggg ttatttcttc gacgaccacg tccgcttcat ggatgccttc
     2641 atcgaagccc tgggtctgga agaggtcgtc ctggtcattc acgactgggg ctccgctctg
     2701 ggtttccact gggccaagcg caatccagag cgcgtcaaag gtattgcatt tatggagttc
     2761 atccgcccta tcccgacctg ggacggcagt ggcagcggta gcgatattcc ggctacatat
     2821 gaatttaccg atggtaaaca ttatatcacc aatgaaccga taccgccgaa aggcagcggc
     2881 agtggtagcg aatggccaga atttgcccgc gagaccttcc aggccttccg caccaccgac
     2941 gtcggccgca agctgatcat cgatcagaac gtttttatcg agggtacgct gccgatgggt
     3001 gtcgtccgcc cgctgactga agtcgagatg gaccattacc gcgagccgtt cctgaatcct
     3061 gttgaccgcg agccactgtg gcgcttccca aacgagctgc caatcgccgg tgagccagcg
     3121 aacatcgtcg cgctggtcga agaatacatg gactggctgc accagtcccc tgtcccgaag
     3181 ctgctgttct ggggcacccc aggcgttctg atcccaccgg ccgaagccgc tcgcctggcc
     3241 aaaagcctgc ctaacagcaa ggctgtggac atcggcccgg gtctgaatct gctgcaagaa
     3301 gacaacccgg acctgatcgg cagcgagatc gcgcgctggc tgtctactct ggagatttcc
     3361 ggttaataaa agcttgcggc cgcactcgag cactaccacc accaccactg agatccggct
     3421 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     3481 taaccccttg gggcctctaa acgggtcttg aggggttttt gctgaaagga ggaactatat
     3541 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3601 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     3661 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3721 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     3781 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     3841 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     3901 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     3961 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     4021 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     4081 atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa
     4141 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     4201 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
     4261 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     4321 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc
     4381 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     4441 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa
     4501 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     4561 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     4621 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     4681 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     4741 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     4801 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     4861 atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat
     4921 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc
     4981 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     5041 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5101 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     5161 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     5221 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5281 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5341 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5401 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5461 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5521 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5581 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5641 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     5701 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     5761 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     5821 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     5881 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     5941 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     6001 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     6061 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     6121 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     6181 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     6241 ttggtcactg atgcctccgt gtaaggggga tttctgttc
//
