LOCUS       sequence-338661-        5751 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 PhoA 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 5751)
  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 5751)
  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-4
FEATURES             Location/Qualifiers
     source          1..5751
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     535..554
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(574..651)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     857..876
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     960..979
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        960..978
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1017..1039
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     sig_peptide     1050..1109
                     /codon_start=1
                     /product="signal sequence of E. coli alkaline phosphatase 
                     (Inouye et al., 1982)"
                     /label=phoA signal sequence
                     /translation="KQSTIALALLPLLFTPVTKA"
     CDS             1224..1526
                     /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     1408..1427
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(1588..1606)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1602..1649
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1686..2141
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1773..1792)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1983..2004
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2233..3048)
                     /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(2233..3048)
                     /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     2956..2975
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3040..3059
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3170..3758
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3659..3678
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    4001..4140
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4066..4085)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4226..4248
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4242..4433)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5242..5751,1..573))
                     /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"
ORIGIN
        1 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
       61 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
      121 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
      181 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
      241 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
      301 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
      361 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
      421 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
      481 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
      541 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
      601 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
      661 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
      721 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
      781 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
      841 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
      901 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt
      961 aatacgactc actatagggg aattgtgagc ggagaacaat tcccctctag aaataatttg
     1021 tttaacttta agaaggagat taacgtatga agcaaagcac tattgcactg gcactcttac
     1081 cgttactgtt tacccctgtg acaaaagccc atcgaagagc tactaaaagc cagataacag
     1141 tatgcgtatt tgcgcgctga tttttgcggt ataagaatat atactgatat gtatacccga
     1201 agtatgtcaa aaagaggtat gctatgcagt ttaaggttta cacctataaa agagagagcc
     1261 gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg cgacggatgg
     1321 tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg
     1381 tggtgcatat cggggataaa agctggcgca tgatgaccac cgatatggcc agtgtgccgg
     1441 tctccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg
     1501 ccattaacct gatgttctgg ggaataggct cttcgtaaga tccggctgct aacaaagccc
     1561 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     1621 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     1681 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     1741 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     1801 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     1861 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     1921 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     1981 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     2041 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     2101 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     2161 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     2221 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     2281 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     2341 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     2401 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     2461 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     2521 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     2581 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     2641 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     2701 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     2761 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     2821 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     2881 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     2941 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     3001 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt
     3061 attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt
     3121 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     3181 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     3241 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     3301 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     3361 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     3421 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     3481 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     3541 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     3601 acaggtatcc ggtaagcggc agggtcggaa caggaaagcg cacgagggag cttccagggg
     3661 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     3721 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     3781 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     3841 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     3901 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gggcctgatg cggtattttc
     3961 tccttacgca tctgtcagtg agcgaggaag cggaagaggg cctgatgcgg tattttctcc
     4021 ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga
     4081 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     4141 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     4201 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     4261 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     4321 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     4381 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     4441 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     4501 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     4561 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     4621 ttcgttgata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     4681 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     4741 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     4801 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     4861 cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata
     4921 atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg
     4981 gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag
     5041 cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata
     5101 aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg
     5161 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     5221 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     5281 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     5341 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     5401 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     5461 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     5521 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     5581 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     5641 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     5701 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga c
//
