LOCUS       sequence-129620-        5793 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression plasmid for N-terminal his tagged S.pyogenes SrtA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5793)
  AUTHORS   Guimaraes CP, Witte MD, Theile CS, Bozkurt G, Kundrat L, Blom AE, 
            Ploegh HL
  TITLE     Site-specific C-terminal and internal loop labeling of proteins 
            using sortase-mediated reactions.
  JOURNAL   Nat Protoc. 2013 Sep;8(9):1787-99. doi: 10.1038/nprot.2013.101. Epub
            2013 Aug 29.
  PUBMED    23989673
REFERENCE   2  (bases 1 to 5793)
  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: 
            "10.1038/nprot.2013.101"; journalName: "Nat Protoc"; date: 
            "2013-09"; volume: "8"; issue: "9"; pages: "1787-99"
COMMENT     Sequence Label: pET28a-SpySrtA
FEATURES             Location/Qualifiers
     source          1..5793
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    5..144
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(70..89)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     230..252
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(246..437)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1246..2328)
                     /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     2290..2309
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2329..2406)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2612..2631
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2715..2734
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2715..2733
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2734..2758
                     /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             2773..2795
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2814..2831
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2841..2858
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3372..3389
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3442..3460)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3456..3503
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3540..3995
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3627..3646)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3837..3858
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4087..4902)
                     /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(4087..4902)
                     /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     4810..4829
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4894..4913
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5024..5612
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5513..5532
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5766..5783
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
       61 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
      121 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
      181 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
      241 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
      301 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
      361 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
      421 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
      481 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
      541 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
      601 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
      661 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
      721 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
      781 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
      841 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
      901 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
      961 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     1021 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     1081 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     1141 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     1201 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     1261 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     1321 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     1381 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     1441 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     1501 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     1561 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     1621 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     1681 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     1741 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     1801 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     1861 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     1921 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     1981 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     2041 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     2101 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     2161 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     2221 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     2281 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     2341 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     2401 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     2461 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     2521 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     2581 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     2641 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     2701 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     2761 tctagaaata attttgttta actttaagaa ggagatatac catgcgcagc agccatcatc
     2821 atcatcatca cagcagcggc ctggtgccgc gcggatccgt cttgcaagca caaatggcgg
     2881 ctcagcaact tcctgttata gggggcattg ccataccaga gcttggcatt aatttaccaa
     2941 tttttaaagg tttaggaaat actgagctta tttatggcgc aggaacgatg aaagaagaac
     3001 aagttatggg aggagaaaat aattattctc ttgccagtca tcatattttt ggaattacag
     3061 gttcatctca aatgctcttt tcgccgcttg aaagagcaca aaatgggatg tccatctatt
     3121 taacagataa agaaaaaatt tacgaataca tcataaaaga tgttttcacg gtagctcctg
     3181 aacgcgttga tgttatcgat gatacagctg gtctcaaaga agtgacttta gtgacttgta
     3241 cagatatcga agcaacagaa cgtattattg tcaaaggaga actaaaaaca gaatacgact
     3301 ttgataaagc gcccgccgat gtattgaaag cttttaatca ttcttataac caagtatcta
     3361 cctagctcga gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg
     3421 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     3481 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga
     3541 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3601 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3661 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3721 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     3781 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3841 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     3901 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3961 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     4021 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat
     4081 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     4141 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     4201 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     4261 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     4321 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     4381 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     4441 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
     4501 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     4561 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     4621 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     4681 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     4741 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     4801 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     4861 tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
     4921 gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt
     4981 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     5041 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     5101 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     5161 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     5221 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     5281 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     5341 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     5401 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     5461 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     5521 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     5581 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     5641 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     5701 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     5761 agcgcagcga gtcagtgagc gaggaagcgg aag
//
