LOCUS       sequence-206278-        7209 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Xylanase design 8.1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7209)
  AUTHORS   Lapidoth G, Khersonsky O, Lipsh R, Dym O, Albeck S, Rogotner S, 
            Fleishman SJ
  TITLE     Highly active enzymes by automated combinatorial backbone assembly 
            and sequence design.
  JOURNAL   Nat Commun. 2018 Jul 17;9(1):2780. doi: 10.1038/s41467-018-05205-5.
  PUBMED    30018322
REFERENCE   2  (bases 1 to 7209)
  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"; journalName: "Nat 
            Commun."; date: "2018-07-17"; volume: "9(1):2780. doi"; pages: " 
            10.1038/s41467-018-05205-5"
COMMENT     Sequence Label: xyl8.1
FEATURES             Location/Qualifiers
     source          1..7209
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             101..118
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             119..1216
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKHDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1073..1082
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1190..1213
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1241..1261
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2291..2314
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2377..2395)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2391..2438
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2475..2930
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2562..2581)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2772..2793
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3022..3837)
                     /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(3022..3837)
                     /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     3745..3764
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3829..3848
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3959..4547
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4448..4467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4701..4718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4733..4872
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4798..4817)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4958..4980
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4974..5165)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5737..6819)
                     /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     6781..6800
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6820..6897)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7103..7122
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gggcagcagc catcatcatc atcatcacaa
      121 aatcgaagaa ggtaaactgg taatctggat taacggcgat aaaggctata acggtctcgc
      181 tgaagtcggt aagaaattcg agaaagatac cggaattaaa gtcaccgttg agcatccgga
      241 taaactggaa gagaaattcc cacaggttgc ggcaactggc gatggccctg acattatctt
      301 ctgggcacac gaccgctttg gtggctacgc tcaatctggc ctgttggctg aaatcacccc
      361 ggacaaagcg ttccaggaca agctgtatcc gtttacctgg gatgccgtac gttacaacgg
      421 caagctgatt gcttacccga tcgctgttga agcgttatcg ctgatttata acaaagatct
      481 gctgccgaac ccgccaaaaa cctgggaaga gatcccggcg ctggataaag aactgaaagc
      541 gaaaggtaag agcgcgctga tgttcaacct gcaagaaccg tacttcacct ggccgctgat
      601 tgctgctgac gggggttatg cgttcaagta tgaaaacggc aagcacgaca ttaaagacgt
      661 gggcgtggat aacgctggcg cgaaagcggg tctgaccttc ctggttgacc tgattaaaaa
      721 caaacacatg aatgcagaca ccgattactc catcgcagaa gctgccttta ataaaggcga
      781 aacagcgatg accatcaacg gcccgtgggc atggtccaac atcgacacca gcaaagtgaa
      841 ttatggtgta acggtactgc cgaccttcaa gggtcaacca tccaaaccgt tcgttggcgt
      901 gctgagcgca ggtattaacg ccgccagtcc gaacaaagag ctggcaaaag agttcctcga
      961 aaactatctg ctgactgatg aaggtctgga agcggttaat aaagacaaac cgctgggtgc
     1021 cgtagcgctg aagtcttacg aggaagagtt ggcgaaagat ccacgtattg ccgccaccat
     1081 ggaaaacgcc cagaaaggtg aaatcatgcc gaacatcccg cagatgtccg ctttctggta
     1141 tgccgtgcgt actgcggtga tcaacgccgc cagcggtcgt cagactgtcg atgaagccct
     1201 gaaagacgcg cagactacta gtggttctgg ttccgcgggt gaaaacctgt acttccaggg
     1261 taccattgga tccgaattcc agaaagacat cccggacctg tacaaagttt tcaaagacta
     1321 cttcccgatc ggtaccgcgg ttaacaccga catcgttgaa ggtcgtgacg cggaagcggc
     1381 ggcgctggtt cgtaaacact tcaacatgct gaccgcggaa aacgcgatga aatggatgta
     1441 cctggaaccg cgtgaaggta aattcgactt cgaagacgcg gacaaaatcg ttaacttcgc
     1501 gaagaaacac ggtatgaaaa tccgtggtca caccctggtt tggcactctc agctgccgtc
     1561 ttgggtttct aacatcaccg acaagaacac cctgcgtaaa gttatgcgta accacatcac
     1621 caccgttatg ggtcactaca aaggtaaaat ctacggttgg gacgttgtta acgaagcgct
     1681 gaacgacgac ggtacctacc gtcagtctgt tttctaccag gttatgggtc cggactacat
     1741 cgcggaagcg ctgcgtgcgg cgcacgaagc ggacccggac gcgaaactgt acatcaacga
     1801 ctacaacatc gaaggtatca acgcgaaatc taacgcgctg tacgaactgg ttcgtaaact
     1861 gatcgaacgt ggtgttccga tcgacggtgt tggtttccag gcgcacttct ctggtggttc
     1921 tccgctgtgc tctaacttcc gtgaaaccat caaacgtttc gcggcgctgg gtgttgacgt
     1981 tcagatcacc gaactggaca tgtctatcta cggttggccg ccgcgtccgg cgtacgaaac
     2041 ctacgacgcg atcccggaag agaaattcca gcagcaggcg gaacgtatga agaaactgtt
     2101 cgaaatgttc aaagaaaact ctgactacgt taccggtgtt accttctggg gtgttcgtga
     2161 cgcggactct tggctgggta aaggtaacgc gccgctgctg ttcgactcta acggtcagcc
     2221 gaaacgtgcg ttctgggcgg ttgtttaact gcaggcgagc tccgtcgaca agcttgcggc
     2281 cgcactcgag caccaccacc accaccacca ccactaattg attaatacct aggctgctaa
     2341 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     2401 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     2461 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2521 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2581 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2641 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     2701 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     2761 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     2821 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     2881 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     2941 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3001 tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     3061 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     3121 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     3181 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     3241 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt
     3301 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     3361 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg
     3421 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     3481 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     3541 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     3601 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     3661 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     3721 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     3781 catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac
     3841 accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt
     3901 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     3961 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4021 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4081 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     4141 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4201 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4261 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4321 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4381 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4441 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4501 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     4561 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     4621 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     4681 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     4741 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
     4801 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     4861 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     4921 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     4981 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     5041 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     5101 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     5161 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     5221 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     5281 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     5341 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     5401 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     5461 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     5521 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     5581 cgccagccta gccgggtcct caacgacagg agcacgatca tgctagtcat gccccgcgcc
     5641 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     5701 taatgagtga gctaacttac attaattgcg ttgtgctcac tgcccgcttt ccagtcggga
     5761 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5821 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     5881 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     5941 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     6001 gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     6061 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     6121 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     6181 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     6241 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     6301 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     6361 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     6421 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     6481 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     6541 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     6601 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     6661 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     6721 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     6781 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     6841 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtcctg
     6901 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     6961 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     7021 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     7081 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     7141 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc gatctcgatc
     7201 ccgcgaaat
//
