LOCUS       sequence-205298-        5644 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for recombinant expression in bacteria of SUMO domain fused 
            to DogTag, permitting covalent linkage to SnoopTagJr.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5644)
  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 5644)
  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-SUMO-DogTag
FEATURES             Location/Qualifiers
     source          1..5644
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     563..582
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(602..679)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     885..904
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     988..1007
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        988..1006
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1007..1031
                     /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             1046..1068
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1090..1107
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1405..1473
                     /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             1496..1513
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1566..1584)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1580..1627
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1664..2119
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1751..1770)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1961..1982
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2211..3026)
                     /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(2211..3026)
                     /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     2934..2953
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3018..3037
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3148..3736
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3637..3656
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3890..3907
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3922..4061
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3987..4006)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4147..4169
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4163..4354)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5163..5644,1..601))
                     /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 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
       61 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
      121 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
      181 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
      241 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
      301 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
      361 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
      421 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
      481 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
      541 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
      601 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
      661 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
      721 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
      781 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
      841 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
      901 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
      961 aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
     1021 ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggt
     1081 agctggagcc atcaccatca tcatcacggg agcgatagcg aagtgaacca ggaagcgaaa
     1141 ccggaagtta aaccggaagt gaaaccggaa acccatatta atctgaaagt tagcgacggc
     1201 agcagcgaaa tcttttttaa aattaaaaaa accaccccgc tgcgtcgcct gatggaagcc
     1261 tttgcgaaac gtcagggtaa agaaatggat agcctgcgct ttctgtatga cggcatccgt
     1321 attcaggccg atcagacccc ggaagacctg gatatggaag acaacgatat tattgaagcg
     1381 catcgcgaac agatcggtgg tggcgatatt ccggctacat atgaatttac cgatggtaaa
     1441 cattatatca ccaatgaacc gataccgccg aaataagctt gctaacgcac tcgagcacca
     1501 ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     1561 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     1621 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc
     1681 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     1741 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     1801 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     1861 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     1921 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     1981 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     2041 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     2101 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     2161 gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac
     2221 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     2281 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     2341 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     2401 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     2461 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc
     2521 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     2581 agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
     2641 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     2701 acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta
     2761 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
     2821 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     2881 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
     2941 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa
     3001 gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
     3061 agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     3121 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     3181 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     3241 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     3301 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     3361 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     3421 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     3481 cacacagccc agcttggagc gaacgaccta caccggactg agatacctac agcgtgagct
     3541 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     3601 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     3661 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     3721 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3781 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     3841 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     3901 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3961 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     4021 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     4081 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     4141 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     4201 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     4261 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     4321 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     4381 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     4441 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     4501 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     4561 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     4621 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     4681 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     4741 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     4801 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga
     4861 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     4921 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     4981 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     5041 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     5101 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     5161 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5221 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     5281 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     5341 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     5401 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     5461 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     5521 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     5581 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     5641 tgcc
//
