LOCUS       sequence-331492-        5710 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 MalE 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 5710)
  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 5710)
  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-2
FEATURES             Location/Qualifiers
     source          1..5710
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     86..105
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        86..104
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             144..166
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             366..668
                     /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     550..569
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(730..748)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      744..791
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      828..1283
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(915..934)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1125..1146
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1375..2190)
                     /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(1375..2190)
                     /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     2098..2117
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2182..2201
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2312..2900
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2801..2820
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3054..3071
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3086..3225
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3151..3170)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3311..3333
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3327..3518)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4327..5409)
                     /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
                     EACKAAVHNLLTQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5371..5390
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5410..5487)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(5693..5710,1..2)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
       61 gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggag
      121 aacaattccc ctctagaaat aattttgttt aactttaaga aggagatcgt attatgaaaa
      181 ttaaaacagg tgcacgcatc ctcgcattat ccgcattaac gacgatgatg ttttccgcct
      241 cggctctcgc ccatcgaaga gctactaaaa gccagataac agtatgcgta tttgcgcgct
      301 gatttttgcg gtataagaat atatactgat atgtataccc gaagtatgtc aaaaagaggt
      361 atgctatgca gtttaaggtt tacacctata aaagagagag ccgttatcgt ctgtttgtgg
      421 atgtacagag tgatattatt gacacgcccg ggcgacggat ggtgatcccc ctggccagtg
      481 cacgtctgct gtcagataaa gtctcccgtg aactttaccc ggtggtgcat atcggggata
      541 aaagctggcg catgatgacc accgatatgg ccagtgtgcc ggtctccgtt atcggggaag
      601 aagtggctga tctcagccac cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct
      661 ggggaatagg ctcttcgtaa gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
      721 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
      781 gggatttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
      841 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
      901 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
      961 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     1021 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     1081 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     1141 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     1201 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     1261 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     1321 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
     1381 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     1441 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     1501 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     1561 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     1621 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     1681 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     1741 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     1801 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     1861 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     1921 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     1981 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     2041 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     2101 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     2161 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     2221 agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     2281 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2341 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     2401 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     2461 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     2521 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     2581 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     2641 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     2701 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     2761 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     2821 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     2881 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     2941 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     3001 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     3061 cagtgagcga ggaagcggaa gagggcctga tgcggtattt tctccttacg catctgtgcg
     3121 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     3181 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     3241 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     3301 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     3361 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     3421 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     3481 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     3541 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     3601 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     3661 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     3721 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     3781 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     3841 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     3901 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     3961 aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg
     4021 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     4081 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     4141 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     4201 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     4261 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     4321 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4381 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     4441 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     4501 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     4561 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     4621 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     4681 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     4741 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     4801 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     4861 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     4921 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     4981 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     5041 gatcagccca ctgacgcgtt gcgtgagaag attgtgcacc gccgctttac aggcttcgac
     5101 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     5161 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     5221 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     5281 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     5341 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     5401 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     5461 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     5521 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     5581 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     5641 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     5701 cggcgatata
//
