LOCUS       sequence-466935-        5678 bp    DNA     circular SYN 14-APR-2025
DEFINITION  reprogrammed sortase A variant that recognizes alternative substrate
            LAXTG with high activity and specificity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5678)
  AUTHORS   Dorr BM, Ham HO, An C, Chaikof EL, Liu DR
  TITLE     Reprogramming the specificity of sortase enzymes.
  JOURNAL   Proc Natl Acad Sci U S A. 2014 Sep 16;111(37):13343-8. doi: 
            10.1073/pnas.1411179111. Epub 2014 Sep 3.
  PUBMED    25187567
REFERENCE   2  (bases 1 to 5678)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1073/pnas.1411179111"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2014-09-16- 16"; volume: "111"; issue: "37"; pages: "13343-8"
COMMENT     Sequence Label: eSrtA(2A-9) pET29b
FEATURES             Location/Qualifiers
     source          1..5678
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..19)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(107..126)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        332..409
                     /gene="lacI"
                     /label=lacI promoter
     CDS             410..1492
                     /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(429..448)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2301..2492
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2486..2508)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2594..2733
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2649..2668
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2748..2765)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2919..3507)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2999..3018)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3618..3637)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3629..4444
                     /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             3629..4444
                     /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(3702..3721)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(4536..4991)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4673..4694)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4885..4904
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(5028..5075)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     5071..5089
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(5142..5159)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             5617..5639
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    5654..5678
                     /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(join(5678,1..19))
                     /label=T7
                     /note="T7 promoter, forward primer"
ORIGIN
        1 cctatagtga gtcgtattaa tttcgcggga tcgagatcga tctcgatcct ctacgccgga
       61 cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac
      121 atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg
      181 ggtatggtgg caggccccgt ggccggggga ctgttgggcg ccatctcctt gcatgcacca
      241 ttccttgcgg cggcggtgct caacggcctc aacctactac tgggctgctt cctaatgcag
      301 gagtcgcata agggagagcg tcgagatccc ggacaccatc gaatggcgca aaacctttcg
      361 cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt
      421 aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt
      481 gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga
      541 gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat
      601 tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa
      661 atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt
      721 cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat
      781 taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc
      841 ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga
      901 agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct
      961 gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata
     1021 tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc
     1081 cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt
     1141 tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt
     1201 tggtgcggac atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc
     1261 gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt
     1321 gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt
     1381 gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     1441 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     1501 caattaatgt aagttagctc actcattagg caccgggatc tcgaccgatg cccttgagag
     1561 ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta
     1621 tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc tgggtcattt
     1681 tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg
     1741 gaatcttgca cgccctcgct caagccttcg tcactggtcc cgccaccaaa cgtttcggcg
     1801 agaagcaggc cattatcgcc ggcatggcgg ccccacgggt gcgcatgatc gtgctcctgt
     1861 cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat gaatcaccga
     1921 tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga gcaacaacat
     1981 gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca
     2041 ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat
     2101 ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc cgccgcatcc
     2161 ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa
     2221 cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg aacagaaatc
     2281 ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac atggcccgct
     2341 ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac gcggatgaac
     2401 aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc agctgcctcg
     2461 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     2521 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     2581 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct
     2641 taactatgcg gcatcagagc agattgtact gagagtgcac cattgcggtg tgaaataccg
     2701 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
     2761 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     2821 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     2881 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     2941 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3001 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3061 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     3121 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     3181 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     3241 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     3301 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     3361 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     3421 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     3481 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     3541 gctcagtgga acgaaaactc acgttaaggg attttggtca tgaacaataa aactgtctgc
     3601 ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcttgctc
     3661 taggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga
     3721 taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg atgcgccaga
     3781 gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag
     3841 actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc
     3901 tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc aggtattaga
     3961 agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt
     4021 gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca
     4081 ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa
     4141 tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga
     4201 ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt
     4261 aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat
     4321 cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata
     4381 tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt
     4441 ctaagaatta attcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     4501 gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt
     4561 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     4621 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     4681 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     4741 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     4801 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     4861 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     4921 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     4981 acagggcgcg tcccattcgc caatccggat atagttcctc ctttcagcaa aaaacccctc
     5041 aagacccgtt tagaggcccc aaggggttat gctagttatt gctcagcggt ggcagcagcc
     5101 aactcagctt cctttcgggc tttgttagca gccggatctc agtggtggtg gtggtggtgg
     5161 gatcctttca cttcggtcgc cacaaaaatt ttacgggatt cccacacgcc ggtttcttcg
     5221 ttataatcat cgcaggtcac cagggtcaac tgtttatctt tgccttcctg ttcatccagc
     5281 acttccaccg cgttcggatg caccttacga atgctggtca ttttatatat acgggtttcg
     5341 ttgcccactt taaaatacac catgctgcca ggtttcgccg ctttcaggtt ggtaaactga
     5401 tagttcggac gatcaataaa ggtatggccc gcaatgctaa tgttctgatc atccaggctt
     5461 tcgttttcgt catgaaagca cacgccccgg ttcagctgtt cacgggtcgc cgggcccgga
     5521 tacaccggtt ctttaatatc cgcatccgga atttcaatat aacccgccac tttgctttta
     5581 tctttcggaa tctgcggttt cgcctgcata tgtatatctc cttcttaaag ttaaacaaaa
     5641 ttatttctag aggggaattg ttatccgctc acaattcc
//
