LOCUS       sequence-404707-        5661 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of SnoopLigase2 which directs formation of an 
            isopeptide bond between SnoopTag2 and DogTag2 peptides.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5661)
  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 5661)
  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 SnoopLigase2
FEATURES             Location/Qualifiers
     source          1..5661
                     /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(563..580)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             603..625
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    640..664
                     /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(664..683)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(665..683)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(767..786)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        992..1069
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1070..2152
                     /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(1089..1108)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2960..3151
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3145..3167)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3253..3392
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3308..3327
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3407..3424)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3578..4166)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3658..3677)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4277..4296)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4288..5103
                     /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             4288..5103
                     /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(4361..4380)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5195..5650)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5332..5353)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5544..5563
                     /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 tcacaccact accaccagag ctgtgatgat gatgatgatg gctccagctg cccatatgta
      601 tatctccttc ttaaagttaa acaaaattat ttctagaggg gaattgttat ccgctcacaa
      661 ttcccctata gtgagtcgta ttaatttcgc gggatcgaga tctcgatcct ctacgccgga
      721 cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac
      781 atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg
      841 ggtatggtgg caggccccgt ggccggggga ctgttgggcg ccatctcctt gcatgcacca
      901 ttccttgcgg cggcggtgct caacggcctc aacctactac tgggctgctt cctaatgcag
      961 gagtcgcata agggagagcg tcgagatccc ggacaccatc gaatggcgca aaacctttcg
     1021 cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt
     1081 aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt
     1141 gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga
     1201 gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat
     1261 tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa
     1321 atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt
     1381 cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat
     1441 taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc
     1501 ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga
     1561 agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct
     1621 gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata
     1681 tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc
     1741 cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt
     1801 tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt
     1861 tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc
     1921 gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt
     1981 gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt
     2041 gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     2101 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     2161 caattaatgt aagttagctc actcattagg caccgggatc tcgaccgatg cccttgagag
     2221 ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta
     2281 tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc tgggtcattt
     2341 cggcgaggac cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg cggtattcgg
     2401 aatcttgcac gccctcgctc aagccttcgt cactggtccc gccaccaaac gtttcggcga
     2461 gaagcaggcc attatcgccg gcatggcggc cccacgggtg cgcatgatcg tgctcctgtc
     2521 gttgaggacc cggctaggct ggcggggttg ccttactggt tagcagaatg aatcaccgat
     2581 acgcgagcga acgtgaagcg actgctgctg caaaacgtct gcgacctgag caacaacatg
     2641 aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag cgccctgcac
     2701 cattatgttc cggatctgca tcgcaggatg ctgctggcta ccctgtggaa cacctacatc
     2761 tgtattaacg aagcgctggc attgaccctg agtgattttt ctctggtccc gccgcatcca
     2821 taccgccagt tgtttaccct cacaacgttc cagtaaccgg gcatgttcat catcagtaac
     2881 ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt acccccatga acagaaatcc
     2941 cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt
     3001 tatcagaagc cagacattaa cgcttctgga gaaactcaac gagctggacg cggatgaaca
     3061 ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca gctgcctcgc
     3121 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     3181 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     3241 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt
     3301 aactatgcgg catcagagca gattgtactg agagtgcacc attgcggtgt gaaataccgc
     3361 acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact
     3421 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     3481 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     3541 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     3601 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     3661 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     3721 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     3781 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     3841 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     3901 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     3961 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     4021 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     4081 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     4141 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     4201 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gaacaataaa actgtctgct
     4261 tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac gtcttgctct
     4321 aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat
     4381 aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag
     4441 ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga
     4501 ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct
     4561 gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca ggtattagaa
     4621 gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg
     4681 cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag
     4741 gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat
     4801 ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat
     4861 tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta
     4921 ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc
     4981 ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat
     5041 ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc
     5101 taagaattaa ttcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     5161 ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta
     5221 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     5281 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     5341 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     5401 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     5461 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     5521 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     5581 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     5641 cagggcgcgt cccattcgcc a
//
