LOCUS       sequence-473516-        5678 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression plasmid for C-terminal his tagged S. aureus SrtA with 
            enhanced catalytic activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5678)
  AUTHORS   Shi J, Kundrat L, Pishesha N, Bilate A, Theile C, Maruyama T, Dougan
            SK, Ploegh HL, Lodish HF
  TITLE     Engineered red blood cells as carriers for systemic delivery of a 
            wide array of functional probes.
  JOURNAL   Proc Natl Acad Sci U S A. 2014 Jul 15;111(28):10131-6. doi: 
            10.1073/pnas.1409861111. Epub 2014 Jun 30.
  PUBMED    24982154
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"; doi: 
            "10.1073/pnas.1409861111"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2014-07-15- 15"; volume: "111"; issue: "28"; pages: "10131-6"
COMMENT     Sequence Label: pet30b-5M SrtA
FEATURES             Location/Qualifiers
     source          1..5678
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     158..177
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(301..348)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     344..362
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(415..432)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             890..912
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    927..951
                     /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)."
     primer_bind     complement(951..970)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(952..970)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1058..1077)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1283..1360
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1361..2443
                     /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     complement(1380..1399)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3252..3443
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3437..3459)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3545..3684
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3600..3619
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3699..3716)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3870..4458)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3950..3969)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4569..4588)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4580..5395
                     /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             4580..5395
                     /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     complement(4653..4672)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(5487..5678,1..264))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5624..5645)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
       61 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
      121 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
      181 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
      241 ttaatgcgcc gctacagggc gcgtcccatt cgccaatccg gatatagttc ctcctttcag
      301 caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt tatgctagtt attgctcagc
      361 ggtggcagca gccaactcag cttcctttcg ggctttgtta gcagccggat ctcagtggtg
      421 gtggtggtgg tgctcgagtt tgacttctgt agctacaaag attttacgtg tttcccaaac
      481 gcctgtctct tcattgtaat catcacaagt aattaatgtt aattgtttat ctttaccttt
      541 ttgttcatct agaacttcta cagctgttgg cttaacgttt cttatacttg tcattttata
      601 cttacgtgtt tcattaccaa ctttaaagta caccatacta ccttttttgg ctgctttaag
      661 atttgtaaat tgatagttcg gacggtcaat gaaagtgtgt cctgcaattg aaatattttg
      721 atcatctagt gattcatttt cttctgcaaa gcttacacct ctatttaatt gttcgcgtgt
      781 tgctggtcct ggatatactg gttctttaat atcagcatct ggaatttcaa tatagcctgc
      841 cacttttgat ttatctttcg gaatttgagg tttagcttgc atatgtatat ctccttctta
      901 aagttaaaca aaattatttc tagaggggaa ttgttatccg ctcacaattc ccctatagtg
      961 agtcgtatta atttcgcggg atcgagatcg atctcgatcc tctacgccgg acgcatcgtg
     1021 gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat
     1081 ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg
     1141 gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg
     1201 gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat
     1261 aagggagagc gtcgagatcc cggacaccat cgaatggcgc aaaacctttc gcggtatggc
     1321 atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata
     1381 cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc
     1441 cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta
     1501 cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc
     1561 cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc
     1621 cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg
     1681 taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc
     1741 gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt
     1801 tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac
     1861 gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg
     1921 cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg
     1981 caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca
     2041 acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga
     2101 tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga
     2161 catctcggta gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac
     2221 caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact
     2281 ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa
     2341 aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     2401 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     2461 taagttagct cactcattag gcaccgggat ctcgaccgat gcccttgaga gccttcaacc
     2521 cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct
     2581 tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg
     2641 accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc
     2701 acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg
     2761 ccattatcgc cggcatggcg gccccacggg tgcgcatgat cgtgctcctg tcgttgagga
     2821 cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc
     2881 gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct
     2941 tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt
     3001 tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa
     3061 cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca
     3121 gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg
     3181 tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca
     3241 cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa
     3301 gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca
     3361 tctgtgaatc gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg
     3421 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     3481 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     3541 ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc
     3601 ggcatcagag cagattgtac tgagagtgca ccattgcggt gtgaaatacc gcacagatgc
     3661 gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc
     3721 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     3781 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     3841 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     3901 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     3961 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4021 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4081 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4141 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4201 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4261 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     4321 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4381 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4441 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4501 aacgaaaact cacgttaagg gattttggtc atgaacaata aaactgtctg cttacataaa
     4561 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcttgct ctaggccgcg
     4621 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     4681 gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct
     4741 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     4801 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
     4861 atggttactc accactgcga tccccgggaa aacagcattc caggtattag aagaatatcc
     4921 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
     4981 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc
     5041 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     5101 tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt
     5161 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     5221 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     5281 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     5341 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaagaatt
     5401 aattcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     5461 gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc
     5521 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     5581 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     5641 tccactatta aagaacgtgg actccaacgt caaagggc
//
