LOCUS       sequence-338663-        6648 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pLyGo cloning vector for a sequence of interest (LPMO) in B. 
            subtilis. Vector encoding the BatLPMO10 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 6648)
  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 6648)
  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-4
FEATURES             Location/Qualifiers
     source          1..6648
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      55..510
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(142..161)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     352..373
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(602..1417)
                     /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(602..1417)
                     /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     1325..1344
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1409..1428
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1539..2127
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2028..2047
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2281..2298
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2366..2505
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2431..2450)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2591..2613
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2607..2798)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     terminator      complement(4315..4401)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    4524..4545
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             5627..5932
                     /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     5811..5830
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     terminator      5976..6070
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
ORIGIN
        1 gttctcttcc ccattgaaga agacttagga actatatccg gattggcgaa tgggacgcgc
       61 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
      121 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
      181 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
      241 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
      301 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
      361 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
      421 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
      481 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
      541 aacctctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
      601 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
      661 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
      721 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
      781 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
      841 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
      901 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
      961 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     1021 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     1081 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     1141 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     1201 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     1261 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     1321 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     1381 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     1441 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     1501 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     1561 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     1621 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     1681 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     1741 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     1801 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     1861 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     1921 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     1981 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     2041 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     2101 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     2161 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     2221 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     2281 agcgagtcag tgagcgagga agcggaagag ggcctgatgc ggtattttct ccttacgcat
     2341 ctgtgagcga ggaagcggaa gagggcctga tgcggtattt tctccttacg catctgtgcg
     2401 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     2461 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     2521 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     2581 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     2641 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     2701 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     2761 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     2821 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     2881 actgatgatg aacatgcact ggcggtatgt ttgcaaaacg attcaaaacc tctttactgc
     2941 cgttattcgc tggattttta ttgctgtttc atttggttct ggcaggaccg gcggctgcga
     3001 gtgctgaaac ggcgaacaaa tcgaatgagc ttacagcacc gtcgatcaaa agcggaacca
     3061 ttcttcatgc atggaattgg tcgttcaata cgttaaaaca caatatgaag gatattcatg
     3121 atgcaggata tacagccatt cagacatctc cgattaacca agtaaaggaa gggaatcaag
     3181 gagataaaag catgtcgaac tggtactggc tgtatcagcc gacatcgtat caaattggca
     3241 accgttactt aggtactgaa caagaattta aagaaatgtg tgcagccgct gaagaatatg
     3301 gcataaaggt cattgttgac gcggtcatca atcataccac cagtgattat gccgcgattt
     3361 ccaatgaggt taagagtatt ccaaactgga cacatggaaa cacacaaatt aaaaactggt
     3421 ctgatcgatc gtctcagcga ggcgcgtcag gttagtgaca ttagaaaacc gactgtaaaa
     3481 agtacagtcg gcattatctc atattataaa agccagtcat taggcctatc tgacaattcc
     3541 tgaatagagt tcataaacaa tcctgcatga taaccatcac aaacagaatg atgtacctgt
     3601 aaagatagcg gtaaatatat tgaattacct ttattaatga attttcctgc tgtaataatg
     3661 ggtagaaggt aattactatt attattgata tttaagttaa acccagtaaa tgaagtccat
     3721 ggaataatag aaagagaaaa agcattttca ggtataggtg ttttgggaaa caatttcccc
     3781 gaaccattat atttctctac atcagaaagg tataaatcat aaaactcttt gaagtcattc
     3841 tttacaggag tccaaatacc agagaatgtt ttagatacac catcaaaaat tgtataaagt
     3901 ggctctaact tatcccaata acctaactct ccgtcgctat tgtaaccagt tctaaaagct
     3961 gtatttgagt ttatcaccct tgtcactaag aaaataaatg cagggtaaaa tttatatcct
     4021 tcttgtttta tgtttcggta taaaacacta atatcaattt ctgtggttat actaaaagtc
     4081 gtttgttggt tcaaataatg attaaatatc tcttttctct tccaattgtc taaatcaatt
     4141 ttattaaagt tcatttgata tgcctcctaa atttttatct aaagtgaatt taggaggctt
     4201 acttgtctgc tttcttcatt agaatcaatc cttttttaaa agtcaatatt actgtaacat
     4261 aaatatatat tttaaaaata tcccacttac gcgcccagct gtctagggcg gcggatttgt
     4321 cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc agtctttcga
     4381 ctgagccttt cgttttattt gatgccttta attaagaagc ggagcgcgaa taaaccgcct
     4441 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     4501 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     4561 ttacacttta tgcgtctatt cttttggaag aaaatatagg gaaaatggta tttgttaaaa
     4621 attctgaata tttatacaat atcatatgtt tcacattgaa atatgctgtc cagactgtcc
     4681 gctgtgtaaa aaataggaat aaaggggggt tgttattatt ttactgatat gtaaaatata
     4741 atttgtataa gaaaattcga aacgtaagat gaaaccttag ataaaagtgc tttttttgtt
     4801 gcaattgaag aattattaat gttaagctta attaaagata atatctttga attgtaacgc
     4861 ccctcaaaag taagaactac aaaaaaagaa tacgttatat agaaatatgt ttgaaccttc
     4921 ttcagattac aaatatattc ggacggactc tacctcaaat gcttatctaa ctatagaatg
     4981 acatacaagc acaaccttga aaatttgaaa atataactac caatgaactt gttcatgtga
     5041 attatcgctg tatttaattt tctcaattca atatataata tgccaataca ttgttacaag
     5101 tagaaattaa gacacccttg atagccttac tatacctaac atgatgtagt attaaatgaa
     5161 tatgtaaata tatttatgat aagaagcgac ttatttataa tcattacata tttttctatt
     5221 ggaatgatta agattccaat agaatagtgt ataaattatt tatcttgaaa ggagggatgc
     5281 ctaaaaacga agaacattaa aaacatatat ttgcaccgtc taatggattt atgaaaaatc
     5341 attttatcag tttgaaaatt atgtattatg attaactaat aaggaggaca aacatgaagg
     5401 gttttatcag aggcgttgtt ctaactgctg ttttaggtgt tggtggtact ttatttgcag
     5461 gtgatgtatc tgcacatcga agagcttaac gcaaaaaacc ccgcttcggc ggggtttttt
     5521 cgctactaaa agccagataa cagtatgcgt atttgcgcgc tgatttttgc ggtataagaa
     5581 tatatactga tatgtatacc cgaagtatgt caaaaagagg tatgctatgc agtttaaggt
     5641 ttacacctat aaaagagaga gccgttatcg tctgtttgtg gatgtacaga gtgatattat
     5701 tgacacgccc gggcgacgga tggtgatccc cctggccagt gcacgtctgc tgtcagataa
     5761 agtctcccgt gaactttacc cggtggtgca tatcggggat aaaagctggc gcatgatgac
     5821 caccgatatg gccagtgtgc cggtctccgt tatcggggaa gaagtggctg atctcagcca
     5881 ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc tggggaatat aagctcttcg
     5941 taagatccgg ctgttcagcg tctgaactag tcttggactc ctgttgatag atccagtaat
     6001 gacctcagaa ctccatctgg atttgttcag aacgctcggt tgccgccggg cgttttttat
     6061 tggtgagaat ccaggggtcc ccaataatta cgattgcgcc tcatgttttc cttgagaatt
     6121 acaaaacagg tgtaacacat tctttcaatg atcaactgac gattaccttg cgtgcagatg
     6181 cgaatacaac aaaagccgtt tatcaaatca ataatggacc agagacggcg tttaaggatg
     6241 gagatcaatt cacaatcgga aaaggagatc catttggcaa aacatacacc atcatgttaa
     6301 aaggaacgaa cagtgatggt gtaacgagga ccgagaaata cagttttgtt aaaagagatc
     6361 cagcgtcggc caaaaccatc ggctatcaaa atccgaatca ttggagccag gtaaatgctt
     6421 atatctataa acatgatggg agccgagtaa ttgaattgac cggatcttgg cctggaaaac
     6481 caatgactaa aaatgcagac ggaatttaca cgctgacgct gcctgcggac acggatacaa
     6541 ccaacgcaaa agtgattttt aataatggca gcgcccaagt gcccggtcag aatcagcctg
     6601 gctttgatta cgtgctaaat ggtttatata atgactcggg cttaagcg
//
