LOCUS       sequence-332139-        6607 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pLyGo cloning vector for a sequence of interest (LPMO) in Bacillus 
            subtilis. Vector encoding the AmyQ 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 6607)
  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 6607)
  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-2
FEATURES             Location/Qualifiers
     source          1..6607
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      10..465
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(97..116)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     307..328
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(557..1372)
                     /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(557..1372)
                     /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     1280..1299
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1364..1383
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1494..2082
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1983..2002
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2236..2253
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2268..2407
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2333..2352)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2493..2515
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2509..2700)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     terminator      complement(4217..4303)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    4426..4447
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             5541..5846
                     /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     5725..5744
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     terminator      5890..5984
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
ORIGIN
        1 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
       61 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
      121 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
      181 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
      241 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
      301 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
      361 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
      421 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
      481 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
      541 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
      601 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
      661 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
      721 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
      781 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
      841 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
      901 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
      961 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     1021 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     1081 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     1141 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     1201 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     1261 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     1321 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc
     1381 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt
     1441 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     1501 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     1561 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     1621 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     1681 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     1741 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     1801 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     1861 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     1921 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     1981 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     2041 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     2101 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     2161 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     2221 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagggcct gatgcggtat
     2281 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     2341 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     2401 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     2461 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     2521 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     2581 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     2641 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     2701 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     2761 gaggatgctc acgatacggg ttactgatga tgaacatgca ctggcggtat gtttgcaaaa
     2821 cgattcaaaa cctctttact gccgttattc gctggatttt tattgctgtt tcatttggtt
     2881 ctggcaggac cggcggctgc gagtgctgaa acggcgaaca aatcgaatga gcttacagca
     2941 ccgtcgatca aaagcggaac cattcttcat gcatggaatt ggtcgttcaa tacgttaaaa
     3001 cacaatatga aggatattca tgatgcagga tatacagcca ttcagacatc tccgattaac
     3061 caagtaaagg aagggaatca aggagataaa agcatgtcga actggtactg gctgtatcag
     3121 ccgacatcgt atcaaattgg caaccgttac ttaggtactg aacaagaatt taaagaaatg
     3181 tgtgcagccg ctgaagaata tggcataaag gtcattgttg acgcggtcat caatcatacc
     3241 accagtgatt atgccgcgat ttccaatgag gttaagagta ttccaaactg gacacatgga
     3301 aacacacaaa ttaaaaactg gtctgatcga tcgtctcagc gaggcgcgtc aggttagtga
     3361 cattagaaaa ccgactgtaa aaagtacagt cggcattatc tcatattata aaagccagtc
     3421 attaggccta tctgacaatt cctgaataga gttcataaac aatcctgcat gataaccatc
     3481 acaaacagaa tgatgtacct gtaaagatag cggtaaatat attgaattac ctttattaat
     3541 gaattttcct gctgtaataa tgggtagaag gtaattacta ttattattga tatttaagtt
     3601 aaacccagta aatgaagtcc atggaataat agaaagagaa aaagcatttt caggtatagg
     3661 tgttttggga aacaatttcc ccgaaccatt atatttctct acatcagaaa ggtataaatc
     3721 ataaaactct ttgaagtcat tctttacagg agtccaaata ccagagaatg ttttagatac
     3781 accatcaaaa attgtataaa gtggctctaa cttatcccaa taacctaact ctccgtcgct
     3841 attgtaacca gttctaaaag ctgtatttga gtttatcacc cttgtcacta agaaaataaa
     3901 tgcagggtaa aatttatatc cttcttgttt tatgtttcgg tataaaacac taatatcaat
     3961 ttctgtggtt atactaaaag tcgtttgttg gttcaaataa tgattaaata tctcttttct
     4021 cttccaattg tctaaatcaa ttttattaaa gttcatttga tatgcctcct aaatttttat
     4081 ctaaagtgaa tttaggaggc ttacttgtct gctttcttca ttagaatcaa tcctttttta
     4141 aaagtcaata ttactgtaac ataaatatat attttaaaaa tatcccactt acgcgcccag
     4201 ctgtctaggg cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa
     4261 aacgaaaggc ccagtctttc gactgagcct ttcgttttat ttgatgcctt taattaagaa
     4321 gcggagcgcg aataaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     4381 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     4441 cactcattag gcaccccagg ctttacactt tatgcgtcta ttcttttgga agaaaatata
     4501 gggaaaatgg tatttgttaa aaattctgaa tatttataca atatcatatg tttcacattg
     4561 aaatatgctg tccagactgt ccgctgtgta aaaaatagga ataaaggggg gttgttatta
     4621 ttttactgat atgtaaaata taatttgtat aagaaaattc gaaacgtaag atgaaacctt
     4681 agataaaagt gctttttttg ttgcaattga agaattatta atgttaagct taattaaaga
     4741 taatatcttt gaattgtaac gcccctcaaa agtaagaact acaaaaaaag aatacgttat
     4801 atagaaatat gtttgaacct tcttcagatt acaaatatat tcggacggac tctacctcaa
     4861 atgcttatct aactatagaa tgacatacaa gcacaacctt gaaaatttga aaatataact
     4921 accaatgaac ttgttcatgt gaattatcgc tgtatttaat tttctcaatt caatatataa
     4981 tatgccaata cattgttaca agtagaaatt aagacaccct tgatagcctt actataccta
     5041 acatgatgta gtattaaatg aatatgtaaa tatatttatg ataagaagcg acttatttat
     5101 aatcattaca tatttttcta ttggaatgat taagattcca atagaatagt gtataaatta
     5161 tttatcttga aaggagggat gcctaaaaac gaagaacatt aaaaacatat atttgcaccg
     5221 tctaatggat ttatgaaaaa tcattttatc agtttgaaaa ttatgtatta tgattaacta
     5281 ataaggagga caaacatgat tcaaaaacga aagcggacag tttcgttcag acttgtgctt
     5341 atgtgcacgc tgttatttgt cagtttgccg attacaaaaa catcagccca tcgaagagct
     5401 taacgcaaaa aaccccgctt cggcggggtt ttttcgctac taaaagccag ataacagtat
     5461 gcgtatttgc gcgctgattt ttgcggtata agaatatata ctgatatgta tacccgaagt
     5521 atgtcaaaaa gaggtatgct atgcagttta aggtttacac ctataaaaga gagagccgtt
     5581 atcgtctgtt tgtggatgta cagagtgata ttattgacac gcccgggcga cggatggtga
     5641 tccccctggc cagtgcacgt ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg
     5701 tgcatatcgg ggataaaagc tggcgcatga tgaccaccga tatggccagt gtgccggtct
     5761 ccgttatcgg ggaagaagtg gctgatctca gccaccgcga aaatgacatc aaaaacgcca
     5821 ttaacctgat gttctgggga atataagctc ttcgtaagat ccggctgttc agcgtctgaa
     5881 ctagtcttgg actcctgttg atagatccag taatgacctc agaactccat ctggatttgt
     5941 tcagaacgct cggttgccgc cgggcgtttt ttattggtga gaatccaggg gtccccaata
     6001 attacgattg cgcctcatgt tttccttgag aattacaaaa caggtgtaac acattctttc
     6061 aatgatcaac tgacgattac cttgcgtgca gatgcgaata caacaaaagc cgtttatcaa
     6121 atcaataatg gaccagagac ggcgtttaag gatggagatc aattcacaat cggaaaagga
     6181 gatccatttg gcaaaacata caccatcatg ttaaaaggaa cgaacagtga tggtgtaacg
     6241 aggaccgaga aatacagttt tgttaaaaga gatccagcgt cggccaaaac catcggctat
     6301 caaaatccga atcattggag ccaggtaaat gcttatatct ataaacatga tgggagccga
     6361 gtaattgaat tgaccggatc ttggcctgga aaaccaatga ctaaaaatgc agacggaatt
     6421 tacacgctga cgctgcctgc ggacacggat acaaccaacg caaaagtgat ttttaataat
     6481 ggcagcgccc aagtgcccgg tcagaatcag cctggctttg attacgtgct aaatggttta
     6541 tataatgact cgggcttaag cggttctctt ccccattgaa gaagacttag gaactatatc
     6601 cggattg
//
