LOCUS       sequence-332141-        6598 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pLyGo cloning vector for a sequence of interest (LPMO) in B. 
            subtilis. Vector encoding the AprE signal peptide and the LyGo 
            cassette (SapI-ccdB-SapI).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6598)
  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 6598)
  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-Bs-3
FEATURES             Location/Qualifiers
     source          1..6598
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      50..505
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(137..156)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     347..368
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(597..1412)
                     /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(597..1412)
                     /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     1320..1339
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1404..1423
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1534..2122
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2023..2042
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2276..2293
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2308..2447
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2373..2392)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2533..2555
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2549..2740)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     terminator      complement(4257..4343)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    4466..4487
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             5572..5877
                     /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     5756..5775
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     terminator      5921..6015
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
ORIGIN
        1 cttccccatt gaagaagact taggaactat atccggattg gcgaatggga cgcgccctgt
       61 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
      121 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
      181 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
      241 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
      301 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
      361 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
      421 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
      481 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
      541 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag
      601 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
      661 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
      721 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
      781 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
      841 tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca
      901 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
      961 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     1021 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     1081 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca
     1141 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     1201 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     1261 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
     1321 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta
     1381 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
     1441 gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     1501 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     1561 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     1621 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     1681 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     1741 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     1801 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     1861 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     1921 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     1981 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     2041 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     2101 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     2161 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     2221 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     2281 gtcagtgagc gaggaagcgg aagagggcct gatgcggtat tttctcctta cgcatctgtg
     2341 cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     2401 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
     2461 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     2521 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     2581 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct
     2641 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa
     2701 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg
     2761 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg
     2821 ttactgatga tgaacatgca ctggcggtat gtttgcaaaa cgattcaaaa cctctttact
     2881 gccgttattc gctggatttt tattgctgtt tcatttggtt ctggcaggac cggcggctgc
     2941 gagtgctgaa acggcgaaca aatcgaatga gcttacagca ccgtcgatca aaagcggaac
     3001 cattcttcat gcatggaatt ggtcgttcaa tacgttaaaa cacaatatga aggatattca
     3061 tgatgcagga tatacagcca ttcagacatc tccgattaac caagtaaagg aagggaatca
     3121 aggagataaa agcatgtcga actggtactg gctgtatcag ccgacatcgt atcaaattgg
     3181 caaccgttac ttaggtactg aacaagaatt taaagaaatg tgtgcagccg ctgaagaata
     3241 tggcataaag gtcattgttg acgcggtcat caatcatacc accagtgatt atgccgcgat
     3301 ttccaatgag gttaagagta ttccaaactg gacacatgga aacacacaaa ttaaaaactg
     3361 gtctgatcga tcgtctcagc gaggcgcgtc aggttagtga cattagaaaa ccgactgtaa
     3421 aaagtacagt cggcattatc tcatattata aaagccagtc attaggccta tctgacaatt
     3481 cctgaataga gttcataaac aatcctgcat gataaccatc acaaacagaa tgatgtacct
     3541 gtaaagatag cggtaaatat attgaattac ctttattaat gaattttcct gctgtaataa
     3601 tgggtagaag gtaattacta ttattattga tatttaagtt aaacccagta aatgaagtcc
     3661 atggaataat agaaagagaa aaagcatttt caggtatagg tgttttggga aacaatttcc
     3721 ccgaaccatt atatttctct acatcagaaa ggtataaatc ataaaactct ttgaagtcat
     3781 tctttacagg agtccaaata ccagagaatg ttttagatac accatcaaaa attgtataaa
     3841 gtggctctaa cttatcccaa taacctaact ctccgtcgct attgtaacca gttctaaaag
     3901 ctgtatttga gtttatcacc cttgtcacta agaaaataaa tgcagggtaa aatttatatc
     3961 cttcttgttt tatgtttcgg tataaaacac taatatcaat ttctgtggtt atactaaaag
     4021 tcgtttgttg gttcaaataa tgattaaata tctcttttct cttccaattg tctaaatcaa
     4081 ttttattaaa gttcatttga tatgcctcct aaatttttat ctaaagtgaa tttaggaggc
     4141 ttacttgtct gctttcttca ttagaatcaa tcctttttta aaagtcaata ttactgtaac
     4201 ataaatatat attttaaaaa tatcccactt acgcgcccag ctgtctaggg cggcggattt
     4261 gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtctttc
     4321 gactgagcct ttcgttttat ttgatgcctt taattaagaa gcggagcgcg aataaaccgc
     4381 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     4441 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     4501 ctttacactt tatgcgtcta ttcttttgga agaaaatata gggaaaatgg tatttgttaa
     4561 aaattctgaa tatttataca atatcatatg tttcacattg aaatatgctg tccagactgt
     4621 ccgctgtgta aaaaatagga ataaaggggg gttgttatta ttttactgat atgtaaaata
     4681 taatttgtat aagaaaattc gaaacgtaag atgaaacctt agataaaagt gctttttttg
     4741 ttgcaattga agaattatta atgttaagct taattaaaga taatatcttt gaattgtaac
     4801 gcccctcaaa agtaagaact acaaaaaaag aatacgttat atagaaatat gtttgaacct
     4861 tcttcagatt acaaatatat tcggacggac tctacctcaa atgcttatct aactatagaa
     4921 tgacatacaa gcacaacctt gaaaatttga aaatataact accaatgaac ttgttcatgt
     4981 gaattatcgc tgtatttaat tttctcaatt caatatataa tatgccaata cattgttaca
     5041 agtagaaatt aagacaccct tgatagcctt actataccta acatgatgta gtattaaatg
     5101 aatatgtaaa tatatttatg ataagaagcg acttatttat aatcattaca tatttttcta
     5161 ttggaatgat taagattcca atagaatagt gtataaatta tttatcttga aaggagggat
     5221 gcctaaaaac gaagaacatt aaaaacatat atttgcaccg tctaatggat ttatgaaaaa
     5281 tcattttatc agtttgaaaa ttatgtatta tgattaacta ataaggagga caaacatgaa
     5341 gaaaccgttg gggaaaattg tcgcaagcac cgcactactc atttctgttg cttttagttc
     5401 atcgatcgca tcggctgctc atcgaagagc ttaacgcaaa aaaccccgct tcggcggggt
     5461 tttttcgcta ctaaaagcca gataacagta tgcgtatttg cgcgctgatt tttgcggtat
     5521 aagaatatat actgatatgt atacccgaag tatgtcaaaa agaggtatgc tatgcagttt
     5581 aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt acagagtgat
     5641 attattgaca cgcccgggcg acggatggtg atccccctgg ccagtgcacg tctgctgtca
     5701 gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggataaaag ctggcgcatg
     5761 atgaccaccg atatggccag tgtgccggtc tccgttatcg gggaagaagt ggctgatctc
     5821 agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg aatataagct
     5881 cttcgtaaga tccggctgtt cagcgtctga actagtcttg gactcctgtt gatagatcca
     5941 gtaatgacct cagaactcca tctggatttg ttcagaacgc tcggttgccg ccgggcgttt
     6001 tttattggtg agaatccagg ggtccccaat aattacgatt gcgcctcatg ttttccttga
     6061 gaattacaaa acaggtgtaa cacattcttt caatgatcaa ctgacgatta ccttgcgtgc
     6121 agatgcgaat acaacaaaag ccgtttatca aatcaataat ggaccagaga cggcgtttaa
     6181 ggatggagat caattcacaa tcggaaaagg agatccattt ggcaaaacat acaccatcat
     6241 gttaaaagga acgaacagtg atggtgtaac gaggaccgag aaatacagtt ttgttaaaag
     6301 agatccagcg tcggccaaaa ccatcggcta tcaaaatccg aatcattgga gccaggtaaa
     6361 tgcttatatc tataaacatg atgggagccg agtaattgaa ttgaccggat cttggcctgg
     6421 aaaaccaatg actaaaaatg cagacggaat ttacacgctg acgctgcctg cggacacgga
     6481 tacaaccaac gcaaaagtga tttttaataa tggcagcgcc caagtgcccg gtcagaatca
     6541 gcctggcttt gattacgtgc taaatggttt atataatgac tcgggcttaa gcggttct
//
