LOCUS       sequence-338662-        5698 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pLyGo cloning vector for periplasmic expression in E. coli of a 
            sequence of interest (LPMO). Vector encoding PelB 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 5698)
  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 5698)
  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-6
FEATURES             Location/Qualifiers
     source          1..5698
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    14..38
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             53..75
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     sig_peptide     83..148
                     /codon_start=1
                     /gene="pelB (fragment)"
                     /product="leader peptide for secretion"
                     /label=pelB signal sequence
                     /translation="MKYLLPTAAAGLLLLAAQPAMA"
     CDS             263..565
                     /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     447..466
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(627..645)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      641..688
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      725..1180
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(812..831)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1022..1043
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1272..2087)
                     /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(1272..2087)
                     /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     1995..2014
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2079..2098
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2209..2797
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2698..2717
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2951..2968
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2983..3122
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3048..3067)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3208..3230
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3224..3415)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4224..5306)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5268..5287
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5307..5384)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5590..5609
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(5693..5698,1..14)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5693..5698,1..13)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta
       61 actttaagaa ggagatatca ggatgaagta tctgctgccg accgctgctg ctggtctgct
      121 gctcctcgct gcccagccgg cgatggccca tcgaagagct actaaaagcc agataacagt
      181 atgcgtattt gcgcgctgat ttttgcggta taagaatata tactgatatg tatacccgaa
      241 gtatgtcaaa aagaggtatg ctatgcagtt taaggtttac acctataaaa gagagagccg
      301 ttatcgtctg tttgtggatg tacagagtga tattattgac acgcccgggc gacggatggt
      361 gatccccctg gccagtgcac gtctgctgtc agataaagtc tcccgtgaac tttacccggt
      421 ggtgcatatc ggggataaaa gctggcgcat gatgaccacc gatatggcca gtgtgccggt
      481 ctccgttatc ggggaagaag tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc
      541 cattaacctg atgttctggg gaataggctc ttcgtaagat ccggctgcta acaaagcccg
      601 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
      661 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
      721 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
      781 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
      841 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
      901 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
      961 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     1021 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     1081 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     1141 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     1201 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     1261 tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     1321 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     1381 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     1441 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     1501 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
     1561 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     1621 attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
     1681 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     1741 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     1801 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     1861 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     1921 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     1981 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     2041 tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta
     2101 ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt
     2161 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     2221 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     2281 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     2341 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     2401 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     2461 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     2521 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     2581 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     2641 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     2701 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     2761 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     2821 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     2881 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     2941 gaccgagcgc agcgagtcag tgagcgagga agcggaagag ggcctgatgc ggtattttct
     3001 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
     3061 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     3121 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3181 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3241 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     3301 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     3361 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     3421 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     3481 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     3541 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     3601 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     3661 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     3721 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     3781 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     3841 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga
     3901 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     3961 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     4021 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     4081 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     4141 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     4201 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4261 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     4321 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     4381 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     4441 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     4501 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     4561 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     4621 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     4681 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     4741 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     4801 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     4861 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     4921 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     4981 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     5041 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     5101 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     5161 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     5221 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     5281 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     5341 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     5401 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     5461 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     5521 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     5581 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     5641 gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatac
//
