LOCUS       sequence-331491-        5926 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pLyGo cloning vector for cytoplasmic expression in E. coli of a 
            sequence of interest (LPMO). Vector encoding Ubiquitin with an 
            internal His10-tag and the LyGo cassette (SapI-ccdB-SapI).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5926)
  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 5926)
  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-1
FEATURES             Location/Qualifiers
     source          1..5926
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      69..524
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(156..175)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     366..387
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(616..1431)
                     /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(616..1431)
                     /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     1339..1358
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1423..1442
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1553..2141
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2042..2061
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2295..2312
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2327..2466
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2392..2411)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2552..2574
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2568..2759)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3568..4650)
                     /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     4612..4631
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4651..4728)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4934..4953
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5041..5060
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5041..5059
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5060..5084
                     /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             5099..5121
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5195..5224
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5570..5872
                     /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     5754..5773
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(5897..5915)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      join(5911..5926,1..32)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
       61 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
      121 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
      181 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
      241 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
      301 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
      361 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
      421 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
      481 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
      541 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
      601 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
      661 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
      721 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
      781 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
      841 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
      901 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
      961 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt
     1021 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     1081 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     1141 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     1201 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     1261 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     1321 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     1381 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct
     1441 tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg
     1501 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     1561 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     1621 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     1681 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     1741 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     1801 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     1861 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     1921 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     1981 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     2041 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     2101 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     2161 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     2221 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     2281 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagggcctg atgcggtatt
     2341 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     2401 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     2461 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     2521 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     2581 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     2641 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     2701 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     2761 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     2821 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     2881 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     2941 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     3001 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     3061 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     3121 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     3181 cctagccggg tcctcaacga caggagcacg atcatgcgca cctgtggggc cgccatgccg
     3241 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     3301 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     3361 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     3421 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     3481 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     3541 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     3601 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     3661 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     3721 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     3781 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     3841 taccgagatg tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     3901 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     3961 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     4021 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     4081 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     4141 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     4201 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     4261 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     4321 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     4381 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     4441 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     4501 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     4561 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     4621 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     4681 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     4741 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     4801 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     4861 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     4921 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     4981 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat cgatctcgat cccgcgaaat
     5041 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
     5101 tgtttaactt taagaaggag atatacatat gcagatcttc gtgaaaacac tgaccggcaa
     5161 aaccattacc ctggaggtgg agcctggcag cgcccatcat catcaccatc accatcacca
     5221 tcatgctggt agcagcgata cgatcgaaaa cgtcaaagcc aaaattcagg acaaagaggg
     5281 cattccgcct gatcaacaac gtctgatctt cgccggaaaa cagctggagg atggccgtac
     5341 actgagtgac tataacattc aagagaaatc gacactggaa ctggttctgg agctgcagaa
     5401 tgaaacccag tcagcaactt ctgacgcttc tggcggtgaa aacctgtact tccaacatcg
     5461 aagagctact aaaagccaga taacagtatg cgtatttgcg cgctgatttt tgcggtataa
     5521 gaatatatac tgatatgtat acccgaagta tgtcaaaaag aggtatgcta tgcagtttaa
     5581 ggtttacacc tataaaagag agagccgtta tcgtctgttt gtggatgtac agagtgatat
     5641 tattgacacg cccgggcgac ggatggtgat ccccctggcc agtgcacgtc tgctgtcaga
     5701 taaagtctcc cgtgaacttt acccggtggt gcatatcggg gataaaagct ggcgcatgat
     5761 gaccaccgat atggccagtg tgccggtctc cgttatcggg gaagaagtgg ctgatctcag
     5821 ccaccgcgaa aatgacatca aaaacgccat taacctgatg ttctggggaa taggctcttc
     5881 gtaagatccg gctccaccgc tgagcaataa ctagcataac cccttg
//
