LOCUS       sequence-329722-        5860 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 DsBA 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 5860)
  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 5860)
  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-5
FEATURES             Location/Qualifiers
     source          1..5860
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     72..91
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        72..90
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             130..152
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             331..633
                     /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     515..534
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(695..713)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      709..756
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      793..1248
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(880..899)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1090..1111
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1340..2155)
                     /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(1340..2155)
                     /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     2063..2082
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2147..2166
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2277..2865
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2766..2785
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    3222..3361
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3287..3306)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3447..3469
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3463..3654)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4463..5545)
                     /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     5507..5526
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5546..5623)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5829..5848
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga
       61 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggagaaca attcccctct
      121 agaaataatt ttgtttaact ttaagaagga gatcgtagga tgaaaaagat ttggctggcg
      181 ctggctggtt tagttttagc gtttagcgca tcggcgcatc gaagagctac taaaagccag
      241 ataacagtat gcgtatttgc gcgctgattt ttgcggtata agaatatata ctgatatgta
      301 tacccgaagt atgtcaaaaa gaggtatgct atgcagttta aggtttacac ctataaaaga
      361 gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac gcccgggcga
      421 cggatggtga tccccctggc cagtgcacgt ctgctgtcag ataaagtctc ccgtgaactt
      481 tacccggtgg tgcatatcgg ggataaaagc tggcgcatga tgaccaccga tatggccagt
      541 gtgccggtct ccgttatcgg ggaagaagtg gctgatctca gccaccgcga aaatgacatc
      601 aaaaacgcca ttaacctgat gttctgggga ataggctctt cgtaagatcc ggctgctaac
      661 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
      721 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
      781 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
      841 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
      901 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
      961 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     1021 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     1081 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     1141 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     1201 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     1261 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     1321 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
     1381 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     1441 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     1501 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     1561 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     1621 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     1681 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     1741 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     1801 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     1861 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     1921 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     1981 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     2041 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     2101 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     2161 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     2221 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     2281 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     2341 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     2401 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     2461 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     2521 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     2581 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     2641 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     2701 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     2761 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     2821 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     2881 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     2941 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     3001 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagaggg cctgatgcgg
     3061 tattttctcc ttacgcatct gtcagtgagc gaggaagcgg aagagggcct gatgcggtat
     3121 tttctcctta cgcatctgtc agtgagcgag gaagcggaag agggcctgat gcggtatttt
     3181 ctccttacgc atctgtcagt gagcgaggaa gcggaagagg gcctgatgcg gtattttctc
     3241 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     3301 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     3361 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     3421 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     3481 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     3541 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     3601 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     3661 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     3721 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     3781 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     3841 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     3901 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     3961 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     4021 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     4081 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
     4141 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     4201 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     4261 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     4321 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     4381 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     4441 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4501 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     4561 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     4621 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     4681 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     4741 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     4801 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     4861 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     4921 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     4981 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     5041 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     5101 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     5161 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     5221 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     5281 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     5341 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     5401 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     5461 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     5521 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     5581 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     5641 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     5701 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     5761 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     5821 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
//
