LOCUS       sequence-202162-        5678 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses 7D sortase A variant in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5678)
  AUTHORS   Jeong HJ, Abhiraman GC, Story CM, Ingram JR, Dougan SK
  TITLE     Generation of Ca2+-independent sortase A mutants with enhanced 
            activity for protein and cell surface labeling.
  JOURNAL   PLoS One. 2017 Dec 4;12(12):e0189068. doi: 
            10.1371/journal.pone.0189068. eCollection 2017.
  PUBMED    29200433
REFERENCE   2  (bases 1 to 5678)
  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: "PLoS One.";
            date: "2017-12-4"; volume: "12(12):e0189068. doi"; pages: " 
            10.1371/journal.pone.0189068. eCollection 2017"
COMMENT     Sequence Label: pET30b-7D-SrtA
FEATURES             Location/Qualifiers
     source          1..5678
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             539..556
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(609..627)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      623..670
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      707..1162
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(794..813)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1004..1025
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1254..2069)
                     /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(1254..2069)
                     /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     1977..1996
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2061..2080
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2191..2779
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2680..2699
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2933..2950
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2965..3104
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3030..3049)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3190..3212
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3206..3397)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4206..5288)
                     /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     5250..5269
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5289..5366)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5572..5591
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaagctaaa cctcaaattc cgaaagataa
      121 atcaaaagtg gcaggctata ttgaaattcc agatgctgat attaaagaac cagtatatcc
      181 aggaccagca acacgcgaac aattaaatag aggtgtaagc tttgcaaaag aaaatcaatc
      241 actagatgat caaaatattt caattgcagg acacactttc attggccgtc cgaactatca
      301 atttacaaat cttaaagcag ccaaaaaagg tagtatggtg tactttaaag ttggtaatga
      361 aacacgtaag tataaaatga caagtataag aaacgttaag ccaacagctg tagaagttct
      421 agatgaacaa aaaggtaaag ataaacaatt aacattaatt acttgtgatg attacaatga
      481 agagacaggc gtttgggaaa cacgtaaaat ctttgtagct acagaagtca aactcgagca
      541 ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
      601 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
      661 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
      721 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
      781 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
      841 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
      901 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
      961 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     1021 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     1081 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     1141 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     1201 tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa
     1261 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     1321 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     1381 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     1441 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     1501 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
     1561 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     1621 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     1681 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     1741 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga
     1801 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     1861 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     1921 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     1981 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag
     2041 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca
     2101 gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     2161 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     2221 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     2281 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     2341 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     2401 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     2461 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     2521 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     2581 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     2641 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     2701 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     2761 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     2821 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     2881 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     2941 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     3001 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     3061 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     3121 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     3181 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     3241 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     3301 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     3361 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     3421 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     3481 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     3541 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     3601 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     3661 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     3721 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     3781 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     3841 ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc
     3901 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     3961 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     4021 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     4081 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     4141 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     4201 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4261 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     4321 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     4381 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     4441 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg
     4501 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     4561 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     4621 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     4681 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     4741 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     4801 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     4861 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     4921 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     4981 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     5041 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     5101 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     5161 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     5221 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     5281 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     5341 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     5401 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg
     5461 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
     5521 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
     5581 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc
     5641 ggcgtagagg atcgagatcg atctcgatcc cgcgaaat
//
