LOCUS       sequence-318034-        5412 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 5412)
  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 5412)
  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..5412
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1..846)
                     /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(920..1756)
                     /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        1902..2020
                     /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(2527..2832)
                     /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(2629..2648)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(3047..3064)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3217..3303
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3395..3422
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(3466..3483)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3637..4225)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3717..3736)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4396..5211)
                     /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(4396..5211)
                     /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     5119..5138
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5212..5316)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5384..5402
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 atctttctgc gaattgagat gacgccactg gctgggcgtc atcccggttt cccgggtaaa
       61 caccaccgaa aaatagttac tatcttcaaa gccacattcg gtcgaaatat cactgattaa
      121 caggcggcta tgctggagaa gatattgcgc atgacacact ctgacctgtc gcagatattg
      181 attgatggtc attccagtct gctggcgaaa ttgctgacgc aaaacgcgct cactgcacga
      241 tgcctcatca caaaatttat ccagcgcaaa gggacttttc aggctagccg ccagccgggt
      301 aatcagctta tccagcaacg tttcgctgga tgttggcggc aacgaatcac tggtgtaacg
      361 atggcgattc agcaacatca ccaactgccc gaacagcaac tcagccattt cgttagcaaa
      421 cggcacatgc tgactacttt catgctcaag ctgaccaata acctgccgcg cctgcgccat
      481 ccccatgcta cctaagcgcc agtgtggttg ccctgcgctg gcgttaaatc ccggaatcgc
      541 cccctgccag tcaagattca gcttcagacg ctccgggcaa taaataatat tctgcaaaac
      601 cagatcgtta acggaagcgt aggagtgttt atcatcagca tgaatgtaaa agagatcgcc
      661 acgggtaatg cgataagggc gatcgttgag tacatgcagg ccattaccgc gccagacaat
      721 caccagctca caaaaatcat gtgtatgttc agcaaagaca tcttgcggat aacggtcagc
      781 cacagcgact gcctgctggt cgctggcaaa aaaatcatct ttgagaagtt ttaactgatg
      841 cgccaccgtg gctacctcgg ccagagaacg aagttgatta ttcgcaatat ggcgtacaaa
      901 tacgttgaga agattcgcgt tattgcagaa agccatcccg tccctggcga atatcacgcg
      961 gtgaccagtt aaactctcgg cgaaaaagcg tcgaaaagtg gttactgtcg ctgaatccac
     1021 agcgataggc gatgtcagta acgctggcct cgctgtggcg tagcagatgt cgggctttca
     1081 tcagtcgcag gcggttcagg tatcgctgag gcgtcagtcc cgtttgctgc ttaagctgcc
     1141 gatgtagcgt acgcagtgaa agagaaaatt gatccgccac ggcatcccaa ttcacctcat
     1201 cggcaaaatg gtcctccagc caggccagaa gcaagttgag acgtgatgcg ctgttttcca
     1261 ggttctcctg caaactgctt ttacgcagca agagcagtaa ttgcataaac aagatctcgc
     1321 gactggcggt cgagggtaaa tcattttccc cttcctgctg ttccatctgt gcaaccagct
     1381 gtcgcacctg ctgcaatacg ctgtggttaa cgcgccagtg agacggatac tgcccatcca
     1441 gctcttgtgg cagcaactga ttcagcccgg cgagaaactg aaatcgatcc ggcgagcgat
     1501 acagcacatt ggtcagacac agattatcgg tatgttcata cagatgccga tcatgatcgc
     1561 gtacgaaaca gaccgtgcca ccggtgatgg tatagggctg cccattaaac acatgaatac
     1621 ccgtgccatg ttcgacaatc acaatttcat gaaaatcatg atgatgttca ggaaaatccg
     1681 cctgcgggag ccggggttct atcgccacgg acgcgttacc agacggaaaa aaatccacac
     1741 tatgtaatac ggtcatactg gcctcctgat gtcgtcaaca cggcgaaata gtaatcacga
     1801 ggtcaggttc ttaccttaaa ttttcgacgg aaaaccacgt aaaaaacgtc gatttttcaa
     1861 gatacagcgt gaattttcag gaaatgcggt gagcatcaca tcaccacaat tcagcaaatt
     1921 gtgaacatca tcacgttcat ctttccctgg ttgccaatgg cccattttct tgtcagtaac
     1981 gagaaggtcg cgaatccagg cgctttttag actggtcgta atgaaattca ggaggaatat
     2041 accatgaaag ctactaaact ggtactgggc gcggtaatcc tgggttctac tctgctggca
     2101 ggttgctcca gcaacgctaa aatcgatcag ggaattaacc cgtatgttgg ctttgaaatg
     2161 ggttacgact ggttaggtcg tatgccgtac aaaggcagcg ttgaaaacgg tgcatacaaa
     2221 gctcagggcg ttcaactgac cgctaaactg ggttacccaa tcactgacga cctggacatc
     2281 tacactcgtc tgggtggcat ggtatggcgt gcagacacta aatccaacgt ttatggtaaa
     2341 aaccacgaca ccggcgtttc tccggtcttc gctggcggtg ttgagtacgc gatcactcct
     2401 gaaatcgcta cccgtctgga ataccagtgg accaacaaca tcggtgacgc acacaccatc
     2461 ggcactcgtc cggacaacgg aggtccgggt atggaaaacc tgtacttcca gcatcgaaga
     2521 gcttaattat attccccaga acatcaggtt aatggcgttt ttgatgtcat tttcgcggtg
     2581 gctgagatca gccacttctt ccccgataac ggagaccggc acactggcca tatcggtggt
     2641 catcatgcgc cagcttttat ccccgatatg caccaccggg taaagttcac gggagacttt
     2701 atctgacagc agacgtgcac tggccagggg gatcaccatc cgtcgcccgg gcgtgtcaat
     2761 aatatcactc tgtacatcca caaacagacg ataacggctc tctcttttat aggtgtaaac
     2821 cttaaactgc atagcatacc tctttttgac atacttcggg tatacatatc agtatatatt
     2881 cttataccgc aaaaatcagc gcgcaaatac gcatactgtt atctggcttt tagtagcgaa
     2941 aaaaccccgc cgaagcgggg ttttttgcgg ctcttcgtaa ccaggataga gtcgacctgc
     3001 aggcatgcaa gcttggctgt tttggcggat gagagaagat tttcagcctg atacagatta
     3061 aatcagaacg cagaagcggt ctgataaaac agaatttgcc tggcggcagt agcgcggtgg
     3121 tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg
     3181 ggtctcccca tgcgagagtg gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg
     3241 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca
     3301 aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga
     3361 cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt
     3421 tttgcgtttc agccaggatt tgcgtattgg gcgctactgt gcttcctcgc tcactgactc
     3481 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     3541 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     3601 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     3661 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     3721 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     3781 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     3841 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     3901 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     3961 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4021 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     4081 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4141 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4201 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4261 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     4321 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     4381 aacttggtct gacagttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     4441 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     4501 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     4561 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     4621 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     4681 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     4741 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt
     4801 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     4861 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     4921 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     4981 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     5041 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     5101 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     5161 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca tactcttcct
     5221 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     5281 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     5341 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     5401 gccctttcgt ct
//
