LOCUS       sequence-109960-        3718 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the E105K/E108A mutant of Staphylococcus aureus sortase A 
            in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3718)
  AUTHORS   Hirakawa H, Ishikawa S, Nagamune T
  TITLE     Ca -independent sortase-A exhibits high selective protein ligation 
            activity in the cytoplasm of E. coli.
  JOURNAL   Biotechnol J. 2015 Apr 10. doi: 10.1002/biot.201500012.
  PUBMED    25864513
REFERENCE   2  (bases 1 to 3718)
  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: "Biotechnol 
            J. 2015 Apr 10. doi: 10.1002/biot.201500012."
COMMENT     Sequence Label: pASKt15C+SrtA2woH
FEATURES             Location/Qualifiers
     source          1..3718
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        36..91
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    42..60
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    72..90
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             130..152
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             162..206
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     rep_origin      754..1182
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(841..860)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1051..1072
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1211..1313
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1314..1973
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1380..1399)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1983..2609
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(2059..2078)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(2880..2897)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3014..3559)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(3094..3113)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccatcgaatg gccagatgat taattcctaa tttttgttga cactctatca ttgatagagt
       61 tattttacca ctccctatca gtgatagaga aaagtgaaat gaatagttcg acaaaaatct
      121 agaaataatt ttgtttaact ttaagaagga gatataccat gaaagaaacc gctgctgcta
      181 aattcgaacg ccagcacatg gacagcccag atctgggtac ccaagctaaa cctcaaattc
      241 cgaaagataa atcgaaagtg gcaggctata ttgaaattcc agatgctgat attaaagaac
      301 cagtatatcc aggaccagca acacctgaac aattaaatag aggtgtaagc tttgcaaagg
      361 aaaatgcctc actagatgat caaaatattt caattgcagg acacactttc attgaccgtc
      421 cgaactatca atttacaaat cttaaagcag ccaaaaaagg tagtatggtg tactttaaag
      481 ttggtaatga aacacgtaag tataaaatga caagtataag agatgttaag cctacagatg
      541 tagaagttct agatgaacaa aaaggtaaag ataaacaatt aacattaatt acttgtgatg
      601 attacaatga aaagacaggc gtttgggaaa aacgtaaaat ctttgtagct acagaagtca
      661 aataagttta aacgacctgt gaagtgaaaa atggcgcaca ttgtgcgaca ttttttttgt
      721 ctgccgttta ccgctactgc gtcacggatc tccacgcgcc ctgtagcggc gcattaagcg
      781 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
      841 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
      901 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
      961 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     1021 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     1081 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1141 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atttggcgaa
     1201 aatgagacgt tgatcggcac gtaagaggtt ccaactttca ccataatgaa ataagatcac
     1261 taccgggcgt attttttgag ttatcgagat tttcaggagc taaggaagct aaaatggaga
     1321 aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg
     1381 aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg
     1441 cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc
     1501 ttgcccgcct gatgaatgct catccggaat ttcgtatggc aatgaaagac ggtgagctgg
     1561 tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt
     1621 catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag
     1681 atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt
     1741 ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata
     1801 tggacaactt cttcgccccc gttttcacta tgggcaaata ttatacgcaa ggcgacaagg
     1861 tgctgatgcc gctggcgatt caggttcatc atgccgtctg tgatggcttc catgtcggca
     1921 gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggcggggcg taataggaat
     1981 taatgatgtc tcgtttagat aaaagtaaag tgattaacag cgcattagag ctgcttaatg
     2041 aggtcggaat cgaaggttta acaacccgta aactcgccca gaagctaggt gtagagcagc
     2101 ctacattgta ttggcatgta aaaaataagc gggctttgct cgacgcctta gccattgaga
     2161 tgttagatag gcaccatact cacttttgcc ctttagaagg ggaaagctgg caagattttt
     2221 tacgtaataa cgctaaaagt tttagatgtg ctttactaag tcatcgcgat ggagcaaaag
     2281 tacatttagg tacacggcct acagaaaaac agtatgaaac tctcgaaaat caattagcct
     2341 ttttatgcca acaaggtttt tcactagaga atgcattata tgcactcagc gcagtggggc
     2401 attttacttt aggttgcgta ttggaagatc aagagcatca agtcgctaaa gaagaaaggg
     2461 aaacacctac tactgatagt atgccgccat tattacgaca agctatcgaa ttatttgatc
     2521 accaaggtgc agagccagcc ttcttattcg gccttgaatt gatcatatgc ggattagaaa
     2581 aacaacttaa atgtgaaagt gggtcttaaa agcagcataa cctttttccg tgatggtaac
     2641 ttcactagtt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     2701 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagcgc tagcggagtg
     2761 tatactggct tactatgttg gcactgatga gggtgtcagt gaagtgcttc atgtggcagg
     2821 agaaaaaagg ctgcaccggt gcgtcagcag aatatgtgat acaggatata ttccgcttcc
     2881 tcgctcactg actcgctacg ctcggtcgtt cgactgcggc gagcggaaat ggcttacgaa
     2941 cggggcggag atttcctgga agatgccagg aagatactta acagggaagt gagagggccg
     3001 cggcaaagcc gtttttccat aggctccgcc cccctgacaa gcatcacgaa atctgacgct
     3061 caaatcagtg gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggcg
     3121 gctccctcgt gcgctctcct gttcctgcct ttcggtttac cggtgtcatt ccgctgttat
     3181 ggccgcgttt gtctcattcc acgcctgaca ctcagttccg ggtaggcagt tcgctccaag
     3241 ctggactgta tgcacgaacc ccccgttcag tccgaccgct gcgccttatc cggtaactat
     3301 cgtcttgagt ccaacccgga aagacatgca aaagcaccac tggcagcagc cactggtaat
     3361 tgatttagag gagttagtct tgaagtcatg cgccggttaa ggctaaactg aaaggacaag
     3421 ttttggtgac tgcgctcctc caagccagtt acctcggttc aaagagttgg tagctcagag
     3481 aaccttcgaa aaaccgccct gcaaggcggt tttttcgttt tcagagcaag agattacgcg
     3541 cagaccaaaa cgatctcaag aagatcatct tattaatcag ataaaatatt tctagatttc
     3601 agtgcaattt atctcttcaa atgtagcacc tgaagtcagc cccatacgat ataagttgta
     3661 attctgcctt tttacggttc ctggcctttt gctggccttt tgctcacatg acccgaca
//
