LOCUS       sequence-332146-        6601 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pLyGo cloning vector for a sequence of interest (LPMO) in Bacillus 
            subtilis. Vector encoding the AmyL signal peptide and the LyGo 
            casette (SapI-ccdB-SapI).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6601)
  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 6601)
  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-1
FEATURES             Location/Qualifiers
     source          1..6601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(77..96)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     287..308
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(537..1352)
                     /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(537..1352)
                     /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     1260..1279
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1344..1363
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1474..2062
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1963..1982
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2216..2233
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2248..2387
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2313..2332)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2473..2495
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2489..2680)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     terminator      complement(4197..4283)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    4406..4427
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             5515..5820
                     /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     5699..5718
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     terminator      5864..5958
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      join(6591..6601,1..445)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
       61 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
      121 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
      181 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
      241 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
      301 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
      361 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
      421 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
      481 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag
      541 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
      601 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
      661 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
      721 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
      781 tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca
      841 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
      901 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
      961 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     1021 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca
     1081 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     1141 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     1201 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
     1261 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta
     1321 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
     1381 gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     1441 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     1501 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     1561 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     1621 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     1681 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     1741 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     1801 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     1861 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     1921 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     1981 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     2041 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     2101 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     2161 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     2221 gtcagtgagc gaggaagcgg aagagggcct gatgcggtat tttctcctta cgcatctgtg
     2281 cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     2341 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
     2401 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     2461 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     2521 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct
     2581 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa
     2641 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg
     2701 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg
     2761 ttactgatga tgaacatgca ctggcggtat gtttgcaaaa cgattcaaaa cctctttact
     2821 gccgttattc gctggatttt tattgctgtt tcatttggtt ctggcaggac cggcggctgc
     2881 gagtgctgaa acggcgaaca aatcgaatga gcttacagca ccgtcgatca aaagcggaac
     2941 cattcttcat gcatggaatt ggtcgttcaa tacgttaaaa cacaatatga aggatattca
     3001 tgatgcagga tatacagcca ttcagacatc tccgattaac caagtaaagg aagggaatca
     3061 aggagataaa agcatgtcga actggtactg gctgtatcag ccgacatcgt atcaaattgg
     3121 caaccgttac ttaggtactg aacaagaatt taaagaaatg tgtgcagccg ctgaagaata
     3181 tggcataaag gtcattgttg acgcggtcat caatcatacc accagtgatt atgccgcgat
     3241 ttccaatgag gttaagagta ttccaaactg gacacatgga aacacacaaa ttaaaaactg
     3301 gtctgatcga tcgtctcagc gaggcgcgtc aggttagtga cattagaaaa ccgactgtaa
     3361 aaagtacagt cggcattatc tcatattata aaagccagtc attaggccta tctgacaatt
     3421 cctgaataga gttcataaac aatcctgcat gataaccatc acaaacagaa tgatgtacct
     3481 gtaaagatag cggtaaatat attgaattac ctttattaat gaattttcct gctgtaataa
     3541 tgggtagaag gtaattacta ttattattga tatttaagtt aaacccagta aatgaagtcc
     3601 atggaataat agaaagagaa aaagcatttt caggtatagg tgttttggga aacaatttcc
     3661 ccgaaccatt atatttctct acatcagaaa ggtataaatc ataaaactct ttgaagtcat
     3721 tctttacagg agtccaaata ccagagaatg ttttagatac accatcaaaa attgtataaa
     3781 gtggctctaa cttatcccaa taacctaact ctccgtcgct attgtaacca gttctaaaag
     3841 ctgtatttga gtttatcacc cttgtcacta agaaaataaa tgcagggtaa aatttatatc
     3901 cttcttgttt tatgtttcgg tataaaacac taatatcaat ttctgtggtt atactaaaag
     3961 tcgtttgttg gttcaaataa tgattaaata tctcttttct cttccaattg tctaaatcaa
     4021 ttttattaaa gttcatttga tatgcctcct aaatttttat ctaaagtgaa tttaggaggc
     4081 ttacttgtct gctttcttca ttagaatcaa tcctttttta aaagtcaata ttactgtaac
     4141 ataaatatat attttaaaaa tatcccactt acgcgcccag ctgtctaggg cggcggattt
     4201 gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtctttc
     4261 gactgagcct ttcgttttat ttgatgcctt taattaagaa gcggagcgcg aataaaccgc
     4321 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     4381 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     4441 ctttacactt tatgcgtcta ttcttttgga agaaaatata gggaaaatgg tatttgttaa
     4501 aaattctgaa tatttataca atatcatatg tttcacattg aaatatgctg tccagactgt
     4561 ccgctgtgta aaaaatagga ataaaggggg gttgttatta ttttactgat atgtaaaata
     4621 taatttgtat aagaaaattc gaaacgtaag atgaaacctt agataaaagt gctttttttg
     4681 ttgcaattga agaattatta atgttaagct taattaaaga taatatcttt gaattgtaac
     4741 gcccctcaaa agtaagaact acaaaaaaag aatacgttat atagaaatat gtttgaacct
     4801 tcttcagatt acaaatatat tcggacggac tctacctcaa atgcttatct aactatagaa
     4861 tgacatacaa gcacaacctt gaaaatttga aaatataact accaatgaac ttgttcatgt
     4921 gaattatcgc tgtatttaat tttctcaatt caatatataa tatgccaata cattgttaca
     4981 agtagaaatt aagacaccct tgatagcctt actataccta acatgatgta gtattaaatg
     5041 aatatgtaaa tatatttatg ataagaagcg acttatttat aatcattaca tatttttcta
     5101 ttggaatgat taagattcca atagaatagt gtataaatta tttatcttga aaggagggat
     5161 gcctaaaaac gaagaacatt aaaaacatat atttgcaccg tctaatggat ttatgaaaaa
     5221 tcattttatc agtttgaaaa ttatgtatta tgattaacta ataaggagga caaacatgaa
     5281 acaacaaaaa cggctttacg cccgattgct gccgctgtta tttgcgctca tcttcttgct
     5341 gcctcattct gcagcagcgg cgcatcgaag agcttaacgc aaaaaacccc gcttcggcgg
     5401 ggttttttcg ctactaaaag ccagataaca gtatgcgtat ttgcgcgctg atttttgcgg
     5461 tataagaata tatactgata tgtatacccg aagtatgtca aaaagaggta tgctatgcag
     5521 tttaaggttt acacctataa aagagagagc cgttatcgtc tgtttgtgga tgtacagagt
     5581 gatattattg acacgcccgg gcgacggatg gtgatccccc tggccagtgc acgtctgctg
     5641 tcagataaag tctcccgtga actttacccg gtggtgcata tcggggataa aagctggcgc
     5701 atgatgacca ccgatatggc cagtgtgccg gtctccgtta tcggggaaga agtggctgat
     5761 ctcagccacc gcgaaaatga catcaaaaac gccattaacc tgatgttctg gggaatataa
     5821 gctcttcgta agatccggct gttcagcgtc tgaactagtc ttggactcct gttgatagat
     5881 ccagtaatga cctcagaact ccatctggat ttgttcagaa cgctcggttg ccgccgggcg
     5941 ttttttattg gtgagaatcc aggggtcccc aataattacg attgcgcctc atgttttcct
     6001 tgagaattac aaaacaggtg taacacattc tttcaatgat caactgacga ttaccttgcg
     6061 tgcagatgcg aatacaacaa aagccgttta tcaaatcaat aatggaccag agacggcgtt
     6121 taaggatgga gatcaattca caatcggaaa aggagatcca tttggcaaaa catacaccat
     6181 catgttaaaa ggaacgaaca gtgatggtgt aacgaggacc gagaaataca gttttgttaa
     6241 aagagatcca gcgtcggcca aaaccatcgg ctatcaaaat ccgaatcatt ggagccaggt
     6301 aaatgcttat atctataaac atgatgggag ccgagtaatt gaattgaccg gatcttggcc
     6361 tggaaaacca atgactaaaa atgcagacgg aatttacacg ctgacgctgc ctgcggacac
     6421 ggatacaacc aacgcaaaag tgatttttaa taatggcagc gcccaagtgc ccggtcagaa
     6481 tcagcctggc tttgattacg tgctaaatgg tttatataat gactcgggct taagcggttc
     6541 tcttccccat tgaagaagac ttaggaacta tatccggatt ggcgaatggg acgcgccctg
     6601 t
//
