LOCUS       sequence-109954-        3772 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the P94R/D160N/D165A/K190E/K196T 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 3772)
  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 3772)
  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+SrtA5
FEATURES             Location/Qualifiers
     source          1..3772
                     /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             165..182
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             192..209
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             216..260
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     rep_origin      808..1236
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(895..914)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1105..1126
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1265..1367
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1368..2027
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1434..1453)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2037..2663
                     /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(2113..2132)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(2934..2951)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3068..3613)
                     /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(3148..3167)
                     /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 gggccaccat catcatcatc
      181 attcttctgg tctggtgcca cgcggttctg gtatgaaaga aaccgctgct gctaaattcg
      241 aacgccagca catggacagc ccagatctgg gtacccaagc taaacctcaa attccgaaag
      301 ataaatcgaa agtggcaggc tatattgaaa ttccagatgc tgatattaaa gaaccagtat
      361 atccaggacc agcaacacgg gaacaattaa atagaggtgt aagctttgca gaagaaaacg
      421 aatcactaga tgatcaaaat atttcaattg caggacacac tttcattgac cgtccgaact
      481 atcaatttac aaatcttaaa gcagccaaaa aaggtagtat ggtgtacttt aaagttggta
      541 atgaaacacg taagtataaa atgacaagta taagaaacgt taagcctaca gccgtagaag
      601 ttctagatga acaaaaaggt aaagataaac aattaacatt aattacttgt gatgattaca
      661 atgaagagac aggcgtttgg gaaacccgta aaatctttgt agctacagaa gtcaaataag
      721 tttaaacgac ctgtgaagtg aaaaatggcg cacattgtgc gacatttttt ttgtctgccg
      781 tttaccgcta ctgcgtcacg gatctccacg cgccctgtag cggcgcatta agcgcggcgg
      841 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
      901 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
      961 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     1021 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     1081 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     1141 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     1201 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatttgg cgaaaatgag
     1261 acgttgatcg gcacgtaaga ggttccaact ttcaccataa tgaaataaga tcactaccgg
     1321 gcgtattttt tgagttatcg agattttcag gagctaagga agctaaaatg gagaaaaaaa
     1381 tcactggata taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat
     1441 ttcagtcagt tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt
     1501 taaagaccgt aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc
     1561 gcctgatgaa tgctcatccg gaatttcgta tggcaatgaa agacggtgag ctggtgatat
     1621 gggatagtgt tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc
     1681 tctggagtga ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg
     1741 cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg
     1801 tctcagccaa tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca
     1861 acttcttcgc ccccgttttc actatgggca aatattatac gcaaggcgac aaggtgctga
     1921 tgccgctggc gattcaggtt catcatgccg tctgtgatgg cttccatgtc ggcagaatgc
     1981 ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg ggcgtaatag gaattaatga
     2041 tgtctcgttt agataaaagt aaagtgatta acagcgcatt agagctgctt aatgaggtcg
     2101 gaatcgaagg tttaacaacc cgtaaactcg cccagaagct aggtgtagag cagcctacat
     2161 tgtattggca tgtaaaaaat aagcgggctt tgctcgacgc cttagccatt gagatgttag
     2221 ataggcacca tactcacttt tgccctttag aaggggaaag ctggcaagat tttttacgta
     2281 ataacgctaa aagttttaga tgtgctttac taagtcatcg cgatggagca aaagtacatt
     2341 taggtacacg gcctacagaa aaacagtatg aaactctcga aaatcaatta gcctttttat
     2401 gccaacaagg tttttcacta gagaatgcat tatatgcact cagcgcagtg gggcatttta
     2461 ctttaggttg cgtattggaa gatcaagagc atcaagtcgc taaagaagaa agggaaacac
     2521 ctactactga tagtatgccg ccattattac gacaagctat cgaattattt gatcaccaag
     2581 gtgcagagcc agccttctta ttcggccttg aattgatcat atgcggatta gaaaaacaac
     2641 ttaaatgtga aagtgggtct taaaagcagc ataacctttt tccgtgatgg taacttcact
     2701 agtttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     2761 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gcgctagcgg agtgtatact
     2821 ggcttactat gttggcactg atgagggtgt cagtgaagtg cttcatgtgg caggagaaaa
     2881 aaggctgcac cggtgcgtca gcagaatatg tgatacagga tatattccgc ttcctcgctc
     2941 actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta cgaacggggc
     3001 ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa
     3061 agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc
     3121 agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggcggctccc
     3181 tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc
     3241 gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac
     3301 tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt
     3361 gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg taattgattt
     3421 agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg
     3481 tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt
     3541 cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc
     3601 aaaacgatct caagaagatc atcttattaa tcagataaaa tatttctaga tttcagtgca
     3661 atttatctct tcaaatgtag cacctgaagt cagccccata cgatataagt tgtaattctg
     3721 cctttttacg gttcctggcc ttttgctggc cttttgctca catgacccga ca
//
