LOCUS       sequence-205296-        5719 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for recombinant bacterial expression of SnoopLigase, a 
            protein that promotes the formation of an isopeptide bond between 2 
            peptide tags (SnoopTagJr and DogTag), with AviTag for biotinylation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5719)
  AUTHORS   Buldun CM, Jean JX, Bedford MR, Howarth M
  TITLE     SnoopLigase Catalyzes Peptide-Peptide Locking and Enables 
            Solid-Phase Conjugate Isolation.
  JOURNAL   J Am Chem Soc. 2018 Feb 28;140(8):3008-3018. doi: 
            10.1021/jacs.7b13237. Epub 2018 Feb 14.
  PUBMED    29402082
REFERENCE   2  (bases 1 to 5719)
  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"; doi: 
            "10.1021/jacs.7b13237"; journalName: "J Am Chem Soc"; date: 
            "2018-02-28- 28"; volume: "140"; issue: "8"; pages: "3008-3018"
COMMENT     Sequence Label: pET28a-AviTag-SnoopLigase
FEATURES             Location/Qualifiers
     source          1..5719
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     939..958
                     /label=LacI-R
                     /note="LacI, reverse primer"
     primer_bind     1262..1281
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1365..1384
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1365..1383
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1384..1408
                     /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             1423..1445
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1464..1481
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1491..1535
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     primer_bind     complement(2017..2035)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2031..2078
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2115..2570
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2202..2221)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2412..2433
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2662..3477)
                     /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(2662..3477)
                     /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     3385..3404
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3469..3488
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3599..4187
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4088..4107
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4341..4358
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4373..4512
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4438..4457)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4598..4620
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4614..4805)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5614..5719,1..977))
                     /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"
ORIGIN
        1 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
       61 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
      121 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
      181 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
      241 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
      301 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
      361 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
      421 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
      481 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
      541 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
      601 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
      661 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
      721 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
      781 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
      841 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
      901 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
      961 aacgttactg gtttcacatt cgccacctct gacttgactc tcttccgggc gctatcatgc
     1021 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     1081 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     1141 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     1201 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     1261 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     1321 cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat
     1381 aggggaattg tgagcggata acaattcccc tctagaaata attttgttta actttaagaa
     1441 ggagatatac catgggcagc agccatcatc atcatcatca cagcagcggc ggcctgaacg
     1501 atatcttcga ggcccagaag atcgagtggc acgagggtag cggtgggtct ggtagtggtg
     1561 tgaataagaa cgataaaaag ccgctgcgtg gtgccgtgtt tagcctgcag aaacagcatc
     1621 ccgactatcc cgatatctat ggcgcgattg atcagaatgg gacctatcaa aatgtgcgta
     1681 ccggcgaaga tggtaaactg acctttaaga atctgagcga tggcaaatat cgcctgtttg
     1741 aaaatagcga acccccgggc tataaaccgg tgcagaataa gccgattgtg gcgtttcaga
     1801 ttgtgaatgg cgaagtgcgt gatgtgacca gcattgtgcc gccgggtgtg ccggctacat
     1861 atgaatttac ctaataaaac ggtaaacatt atatcaccaa tgaaccgata ccgccgaaat
     1921 aataaaagct tgcggccgca ctcgagcact accaccacca ccactgagat ccggctgcta
     1981 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     2041 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     2101 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2161 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2221 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2281 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     2341 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     2401 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     2461 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     2521 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     2581 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     2641 tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     2701 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     2761 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     2821 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     2881 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt
     2941 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     3001 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg
     3061 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     3121 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     3181 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     3241 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     3301 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     3361 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     3421 catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac
     3481 accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt
     3541 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     3601 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     3661 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     3721 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     3781 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     3841 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     3901 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     3961 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4021 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4081 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4141 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     4201 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     4261 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     4321 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     4381 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
     4441 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     4501 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     4561 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     4621 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     4681 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     4741 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     4801 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     4861 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     4921 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     4981 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     5041 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     5101 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     5161 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     5221 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc
     5281 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     5341 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     5401 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     5461 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     5521 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     5581 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5641 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5701 gggcgccagg gtggttttt
//
