LOCUS       sequence-404731-        6655 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of SnoopLigase 2 which catalyzes formation of a
            covalent bond between SnoopTag2 and DogTag2 peptides. HaloTag7 
            allows covalent coupling to HaloLink Magne beads..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6655)
  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 6655)
  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-HaloTag7-SnoopLigase2
FEATURES             Location/Qualifiers
     source          1..6655
                     /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(665..1549)
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRSIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNSKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             complement(1559..1576)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1595..1617
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1632..1656
                     /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(1656..1675)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1657..1675)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1759..1778)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             2063..3145
                     /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(2082..2101)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3954..4145
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4139..4161)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4247..4386
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4302..4321
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4401..4418)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4572..5160)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4652..4671)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5271..5290)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5282..6097
                     /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             5282..6097
                     /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(5355..5374)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6189..6644)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6326..6347)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6538..6557
                     /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 ggtagtgctc gagtgcggcc gcaagctttt
      181 attatttcgg cggtatcggt tcattggtga tataatgttt accgttttat taggtaaatt
      241 catatgtagc cggcacaccc ggcggcacaa tgctggtcac atcacgcact tcgccatcca
      301 caatacgaaa gctcacaatc ggcttattct gcaccggttt atagcccggg ggttcgctat
      361 tttcaatcag gcgatatttg ccatcgctca gattcttaaa ggtcagttta ccatcttcgc
      421 cggtacgcac attttgatag gtcccattct gatcaatcgc gccatagata tcgggatagt
      481 cgggatgctg tttctgcagg ctaaacacgg caccacgcag cggcttttta tcgttcttat
      541 tcaccatatg ggatcccttc tccagctgct ccttcagctt cttggcgttg gccagctcct
      601 cggcggcctg tctctgctcc ttctgcttct gggcctccag ggccttcagg ttggcggggg
      661 atccaccgga aatctccaga gtagacagcc agcgcgcgat ctcgctgccg atcaggtccg
      721 ggttgtcttc ttgcagcaga ttcagacccg ggccgatgtc cacagccttg ctgttaggca
      781 ggcttttggc caggcgagcg gcttcggccg gtgggatcag aacgcctggg gtgccccaga
      841 acagcagctt cgggacaggg gactggtgca gccagtccat gtattcttcg accagcgcga
      901 cgatgttcgc tggctcaccg gcgattggca gctcgtttgg gaagcgccac agtggctcgc
      961 ggtcaacagg attcaggaac ggctcgcggt aatggtccat ctcgacttca gtcagcgggc
     1021 ggacgacacc catcggcagc gtaccctcga taaaaacgtt ctgatcgatg atcagcttgc
     1081 ggccgacgtc ggtggtgcgg aaggcctgga aggtctcgcg ggcaaattct ggccattcgt
     1141 cccaggtcgg gatagggcgg atgaactcca taaatgcaat acctttgacg cgctctggat
     1201 tgcgcttggc ccagtggaaa cccagagcgg agccccagtc gtgaatgacc aggacgacct
     1261 cttccagacc cagggcttcg atgaaggcat ccatgaagcg gacgtggtcg tcgaagaaat
     1321 aacccaggtc tggtttgtcg gatttgccca taccgatcag gtctggagca atgctgcgat
     1381 gggtcggtgc aacatgcggg atgatgttgc gccacacgta ggaggaggtc gggttaccgt
     1441 gcaggaacag cacaggggtg ccatcgcgcg gaccaacatc gacgtagtgc atgcgctcgc
     1501 ccaggacttc cacataatgg gggtcgaatg gaaagccagt accgatttcg ccgctgctgt
     1561 gatgatgatg atgatggctg ctgcccatgg tatatctcct tcttaaagtt aaacaaaatt
     1621 atttctagag gggaattgtt atccgctcac aattccccta tagtgagtcg tattaatttc
     1681 gcgggatcga gatctcgatc ctctacgccg gacgcatcgt ggccggcatc accggcgcca
     1741 caggtgcggt tgctggcgcc tatatcgccg acatcaccga tggggaagat cgggctcgcc
     1801 acttcgggct catgagcgct tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg
     1861 gactgttggg cgccatctcc ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc
     1921 tcaacctact actgggctgc ttcctaatgc aggagtcgca taagggagag cgtcgagatc
     1981 ccggacacca tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag
     2041 agtcaagtca gaggtggcga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc
     2101 cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa
     2161 aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc
     2221 acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct
     2281 gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag
     2341 cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa
     2401 tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc
     2461 cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca
     2521 gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca
     2581 tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc
     2641 ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat
     2701 agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct
     2761 gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc
     2821 aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata
     2881 cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca aacaggattt
     2941 tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt
     3001 gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa
     3061 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     3121 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt
     3181 aggcaccggg atctcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt
     3241 gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg
     3301 taggacaggt gccggcagcg ctctgggtca ttttcggcga ggaccgcttt cgctggagcg
     3361 cgacgatgat cggcctgtcg cttgcggtat tcggaatctt gcacgccctc gctcaagcct
     3421 tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca ggccattatc gccggcatgg
     3481 cggccccacg ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg
     3541 gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgct
     3601 gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt
     3661 aaagtctgga aacgcggaag tcagcgccct gcaccattat gttccggatc tgcatcgcag
     3721 gatgctgctg gctaccctgt ggaacaccta catctgtatt aacgaagcgc tggcattgac
     3781 cctgagtgat ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac
     3841 gttccagtaa ccgggcatgt tcatcatcag taacccgtat cgtgagcatc ctctctcgtt
     3901 tcatcggtat cattaccccc atgaacagaa atccccctta cacggaggca tcagtgacca
     3961 aacaggaaaa aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc
     4021 tggagaaact caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg
     4081 accacgctga tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc
     4141 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
     4201 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc
     4261 agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt
     4321 actgagagtg caccattgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     4381 atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     4441 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     4501 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     4561 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     4621 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     4681 tccctcgtgc gctctcctgt tccgaccatg ccgcttaccg gatacctgtc cgcctttctc
     4741 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     4801 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     4861 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     4921 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     4981 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     5041 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5101 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     5161 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     5221 gggattttgg tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
     5281 tatgagccat attcaacggg aaacgtcttg ctctaggccg cgattaaatt ccaacatgga
     5341 tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat
     5401 ctatcgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag
     5461 cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc
     5521 tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc
     5581 gatccccggg aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat
     5641 tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc
     5701 ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt
     5761 ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa
     5821 agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc
     5881 acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt
     5941 cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc
     6001 tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa
     6061 attgcagttt catttgatgc tcgatgagtt tttctaagaa ttaattcatg agcggataca
     6121 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     6181 tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     6241 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     6301 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     6361 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     6421 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     6481 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     6541 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     6601 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgcca
//
