LOCUS       sequence-332138-        5996 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pLyGo cloning vector for surface display in E. coli of a sequence of
            interest (LPMO). Vector encoding the LppSP-OmpA surface expression 
            anchor and the LyGo cassette (SapI-ccdB-SapI).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5996)
  AUTHORS   Hernandez-Rollan C, Falkenberg KB, Rennig M, Bertelsen AB, Ipsen JO,
            Brander S, Daley DO, Johansen KS, Norholm MHH
  TITLE     LyGo: A Platform for Rapid Screening of Lytic Polysaccharide 
            Monooxygenase Production.
  JOURNAL   ACS Synth Biol. 2021 Apr 16;10(4):897-906. doi: 
            10.1021/acssynbio.1c00034. Epub 2021 Apr 2.
  PUBMED    33797234
REFERENCE   2  (bases 1 to 5996)
  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: 
            "10.1021/acssynbio.1c00034"; journalName: "ACS Synth Biol"; date: 
            "2021-04-16- 16"; volume: "10"; issue: "4"; pages: "897-906"
COMMENT     Sequence Label: pLyGo-Ec-8
FEATURES             Location/Qualifiers
     source          1..5996
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     35..53
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             complement(64..909)
                     /codon_start=1
                     /gene="E. coli rhaR"
                     /product="transcriptional activator of rhaR and rhaS"
                     /label=rhaR
                     /note="needed for rhamnose-dependent activation of the rhaB
                     promoter"
                     /translation="MAHQLKLLKDDFFASDQQAVAVADRYPQDVFAEHTHDFCELVIVW
                     RGNGLHVLNDRPYRITRGDLFYIHADDKHSYASVNDLVLQNIIYCPERLKLNLDWQGAI
                     PGFNASAGQPHWRLGSMGMAQARQVIGQLEHESSQHVPFANEMAELLFGQLVMLLNRHR
                     YTSDSLPPTSSETLLDKLITRLAASLKSPFALDKFCDEASCSERVLRQQFRQQTGMTIN
                     QYLRQVRVCHAQYLLQHSRLLISDISTECGFEDSNYFSVVFTRETGMTPSQWRHLNSQK
                     D"
     CDS             complement(983..1819)
                     /codon_start=1
                     /gene="E. coli rhaS"
                     /product="positive regulator of the rhaB promoter
                     "
                     /label=rhaS
                     /note="activated by L-rhamnose"
                     /translation="MTVLHSVDFFPSGNASVAIEPRLPQADFPEHHHDFHEIVIVEHGT
                     GIHVFNGQPYTITGGTVCFVRDHDRHLYEHTDNLCLTNVLYRSPDRFQFLAGLNQLLPQ
                     ELDGQYPSHWRVNHSVLQQVRQLVAQMEQQEGENDLPSTASREILFMQLLLLLRKSSLQ
                     ENLENSASRLNLLLAWLEDHFADEVNWDAVADQFSLSLRTLHRQLKQQTGLTPQRYLNR
                     LRLMKARHLLRHSEASVTDIAYRCGFSDSNHFSTLFRREFNWSPRDIRQGRDGFLQ"
     promoter        1965..2083
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     CDS             complement(2590..2895)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDKSWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(2692..2711)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     3122..3141
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3467..3484)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3637..3723
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3815..3842
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      complement(4284..4872)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4364..4383)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5043..5858)
                     /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(5043..5858)
                     /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     5766..5785
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5859..5963)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
       61 tctatctttc tgcgaattga gatgacgcca ctggctgggc gtcatcccgg tttcccgggt
      121 aaacaccacc gaaaaatagt tactatcttc aaagccacat tcggtcgaaa tatcactgat
      181 taacaggcgg ctatgctgga gaagatattg cgcatgacac actctgacct gtcgcagata
      241 ttgattgatg gtcattccag tctgctggcg aaattgctga cgcaaaacgc gctcactgca
      301 cgatgcctca tcacaaaatt tatccagcgc aaagggactt ttcaggctag ccgccagccg
      361 ggtaatcagc ttatccagca acgtttcgct ggatgttggc ggcaacgaat cactggtgta
      421 acgatggcga ttcagcaaca tcaccaactg cccgaacagc aactcagcca tttcgttagc
      481 aaacggcaca tgctgactac tttcatgctc aagctgacca ataacctgcc gcgcctgcgc
      541 catccccatg ctacctaagc gccagtgtgg ttgccctgcg ctggcgttaa atcccggaat
      601 cgccccctgc cagtcaagat tcagcttcag acgctccggg caataaataa tattctgcaa
      661 aaccagatcg ttaacggaag cgtaggagtg tttatcatca gcatgaatgt aaaagagatc
      721 gccacgggta atgcgataag ggcgatcgtt gagtacatgc aggccattac cgcgccagac
      781 aatcaccagc tcacaaaaat catgtgtatg ttcagcaaag acatcttgcg gataacggtc
      841 agccacagcg actgcctgct ggtcgctggc aaaaaaatca tctttgagaa gttttaactg
      901 atgcgccacc gtggctacct cggccagaga acgaagttga ttattcgcaa tatggcgtac
      961 aaatacgttg agaagattcg cgttattgca gaaagccatc ccgtccctgg cgaatatcac
     1021 gcggtgacca gttaaactct cggcgaaaaa gcgtcgaaaa gtggttactg tcgctgaatc
     1081 cacagcgata ggcgatgtca gtaacgctgg cctcgctgtg gcgtagcaga tgtcgggctt
     1141 tcatcagtcg caggcggttc aggtatcgct gaggcgtcag tcccgtttgc tgcttaagct
     1201 gccgatgtag cgtacgcagt gaaagagaaa attgatccgc cacggcatcc caattcacct
     1261 catcggcaaa atggtcctcc agccaggcca gaagcaagtt gagacgtgat gcgctgtttt
     1321 ccaggttctc ctgcaaactg cttttacgca gcaagagcag taattgcata aacaagatct
     1381 cgcgactggc ggtcgagggt aaatcatttt ccccttcctg ctgttccatc tgtgcaacca
     1441 gctgtcgcac ctgctgcaat acgctgtggt taacgcgcca gtgagacgga tactgcccat
     1501 ccagctcttg tggcagcaac tgattcagcc cggcgagaaa ctgaaatcga tccggcgagc
     1561 gatacagcac attggtcaga cacagattat cggtatgttc atacagatgc cgatcatgat
     1621 cgcgtacgaa acagaccgtg ccaccggtga tggtataggg ctgcccatta aacacatgaa
     1681 tacccgtgcc atgttcgaca atcacaattt catgaaaatc atgatgatgt tcaggaaaat
     1741 ccgcctgcgg gagccggggt tctatcgcca cggacgcgtt accagacgga aaaaaatcca
     1801 cactatgtaa tacggtcata ctggcctcct gatgtcgtca acacggcgaa atagtaatca
     1861 cgaggtcagg ttcttacctt aaattttcga cggaaaacca cgtaaaaaac gtcgattttt
     1921 caagatacag cgtgaatttt caggaaatgc ggtgagcatc acatcaccac aattcagcaa
     1981 attgtgaaca tcatcacgtt catctttccc tggttgccaa tggcccattt tcttgtcagt
     2041 aacgagaagg tcgcgaatcc aggcgctttt tagactggtc gtaatgaaat tcaggaggaa
     2101 tataccatga aagctactaa actggtactg ggcgcggtaa tcctgggttc tactctgctg
     2161 gcaggttgct ccagcaacgc taaaatcgat cagggaatta acccgtatgt tggctttgaa
     2221 atgggttacg actggttagg tcgtatgccg tacaaaggca gcgttgaaaa cggtgcatac
     2281 aaagctcagg gcgttcaact gaccgctaaa ctgggttacc caatcactga cgacctggac
     2341 atctacactc gtctgggtgg catggtatgg cgtgcagaca ctaaatccaa cgtttatggt
     2401 aaaaaccacg acaccggcgt ttctccggtc ttcgctggcg gtgttgagta cgcgatcact
     2461 cctgaaatcg ctacccgtct ggaataccag tggaccaaca acatcggtga cgcacacacc
     2521 atcggcactc gtccggacaa cggaggtccg ggtatggaaa acctgtactt ccagcatcga
     2581 agagcttaat tatattcccc agaacatcag gttaatggcg tttttgatgt cattttcgcg
     2641 gtggctgaga tcagccactt cttccccgat aacggagacc ggcacactgg ccatatcggt
     2701 ggtcatcatg cgccagcttt tatccccgat atgcaccacc gggtaaagtt cacgggagac
     2761 tttatctgac agcagacgtg cactggccag ggggatcacc atccgtcgcc cgggcgtgtc
     2821 aataatatca ctctgtacat ccacaaacag acgataacgg ctctctcttt tataggtgta
     2881 aaccttaaac tgcatagcat acctcttttt gacatacttc gggtatacat atcagtatat
     2941 attcttatac cgcaaaaatc agcgcgcaaa tacgcatact gttatctggc ttttagtagc
     3001 gaaaaaaccc cgccgaagcg gggttttttg cggctcttcg taaccaggat agagtcgacc
     3061 tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg
     3121 gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg
     3181 aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact
     3241 acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct
     3301 acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac
     3361 agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtaga
     3421 gccaggataa gcttggctgt tttggcggat gagagaagat tttcagcctg atacagatta
     3481 aatcagaacg cagaagcggt ctgataaaac agaatttgcc tggcggcagt agcgcggtgg
     3541 tcccacctga ccccatgccg aactcagaag tgaaacgctg tagcgccgat ggtagtgtgg
     3601 ggtctcccca tgcgagagtg gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg
     3661 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca
     3721 aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga
     3781 cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt
     3841 tttgcgtttc agccaggatt tgcgtattgg gcgctactgt gcttcctcgc tcactgactc
     3901 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     3961 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4021 ggccaggaac cgtaaaaagg ccgcgttgct ggcgttttga tggccttttt gcgtttcagc
     4081 caggatttgc gtattgggcg ctactgtgct tcctcgctca ctgactcgct gcgctcggtc
     4141 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4201 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4261 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4321 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4381 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4441 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4501 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4561 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4621 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4681 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     4741 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4801 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4861 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     4921 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     4981 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5041 agttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     5101 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     5161 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     5221 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     5281 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     5341 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     5401 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     5461 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     5521 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     5581 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     5641 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     5701 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     5761 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     5821 ggcctagagc aagacgtttc ccgttgaata tggctcatac tcttcctttt tcaatattat
     5881 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     5941 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcta
//
