LOCUS       sequence-332101-        5695 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 OmpA 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 5695)
  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 5695)
  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-3
FEATURES             Location/Qualifiers
     source          1..5695
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     19..38
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        19..37
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             77..99
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     sig_peptide     107..169
                     /codon_start=1
                     /product="signal peptide from the E. coli outer membrane 
                     protein OmpA"
                     /label=OmpA signal peptide
                     /translation="MKKTAIAIAVALAGFATVAQA"
     CDS             284..586
                     /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     468..487
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(648..666)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      662..709
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      746..1201
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(833..852)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1043..1064
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1293..2108)
                     /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(1293..2108)
                     /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     2016..2035
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2100..2119
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2230..2818
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2719..2738
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2972..2989
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3004..3143
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3069..3088)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3229..3251
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3245..3436)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4245..5327)
                     /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     5289..5308
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5328..5405)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5611..5630
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gagaacaatt
       61 cccctctaga aataattttg tttaacttta agaaggagat gttcgtatga agaagacagc
      121 tatcgcgatt gcagtggcac tggctggttt cgctaccgta gcgcaggccc atcgaagagc
      181 tactaaaagc cagataacag tatgcgtatt tgcgcgctga tttttgcggt ataagaatat
      241 atactgatat gtatacccga agtatgtcaa aaagaggtat gctatgcagt ttaaggttta
      301 cacctataaa agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga
      361 cacgcccggg cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt
      421 ctcccgtgaa ctttacccgg tggtgcatat cggggataaa agctggcgca tgatgaccac
      481 cgatatggcc agtgtgccgg tctccgttat cggggaagaa gtggctgatc tcagccaccg
      541 cgaaaatgac atcaaaaacg ccattaacct gatgttctgg ggaataggct cttcgtaaga
      601 tccggctgct aacaaagccc gaaaggaaac tgagttggct gctgccaccg ctgagcaata
      661 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
      721 aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
      781 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
      841 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
      901 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
      961 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     1021 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     1081 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     1141 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     1201 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     1261 attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga
     1321 aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt
     1381 aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct
     1441 gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg
     1501 ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta
     1561 tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc
     1621 gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg
     1681 ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc
     1741 gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc
     1801 ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg
     1861 gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca
     1921 ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac
     1981 aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat
     2041 aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata
     2101 tggctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac
     2161 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     2221 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     2281 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     2341 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     2401 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     2461 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     2521 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     2581 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     2641 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     2701 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     2761 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     2821 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     2881 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     2941 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     3001 gggcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
     3061 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     3121 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     3181 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     3241 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     3301 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     3361 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     3421 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     3481 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     3541 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     3601 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     3661 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     3721 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     3781 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     3841 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
     3901 gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
     3961 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
     4021 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
     4081 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     4141 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     4201 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     4261 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4321 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     4381 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     4441 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     4501 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
     4561 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     4621 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     4681 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     4741 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     4801 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     4861 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     4921 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     4981 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     5041 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     5101 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     5161 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     5221 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     5281 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     5341 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     5401 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     5461 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     5521 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     5581 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     5641 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgag
//
