LOCUS       sequence-206279-        7242 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Xylanase design 8.3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7242)
  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 7242)
  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: xyl8.3
FEATURES             Location/Qualifiers
     source          1..7242
                     /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             2324..2347
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2410..2428)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2424..2471
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2508..2963
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2595..2614)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2805..2826
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3055..3870)
                     /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(3055..3870)
                     /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     3778..3797
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3862..3881
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3992..4580
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4481..4500
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4734..4751
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4766..4905
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4831..4850)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4991..5013
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5007..5198)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5770..6852)
                     /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     6814..6833
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6853..6930)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7136..7155
                     /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 agaacgacat cccggacctg tactctgttt tcaaagacta
     1321 cttcccgatc ggtgttgcgg ttgacccgtc tcgtctgaac gacaccgacc cgcacgcgca
     1381 gctgaccgcg aaacacttca acatgctgac cgcggaaaac gcgatgaaac cggaatctct
     1441 ggaaccggaa gaaggtcgtt acaacttcga agacgcggac cgtatcgttg cgttcgcgga
     1501 gaaacacggt atgaaaatgc gtggtcacac cctggtttgg caccagcagg ttccggactg
     1561 gttcttcctg gacgaaaacg gtaacccgat ggttgacgaa accgacccga agaaccgtga
     1621 agcgaaccgt gaagaactgc gtcagcgtat ggaaaaccac atcaaaaccg ttgcgggtcg
     1681 ttacaaaggt aaaatctacg cgtgggacgt tgttaacgaa gttttcaacg acgacggtac
     1741 cctgcgtaac tctgcgtggt accagatcat cggtccggac tacatcgaag aagcgctgcg
     1801 tgcggcgcac gaagcggacc cgaacgcgaa actgttcatc aacgactaca acatcgaaaa
     1861 ctggtctcac gcgaaaaccc aggcgatgta caacatggtt cgtgacttca aagaacgtgg
     1921 tgttccggtt gacggtgttg gtatgcaggg tcacatctct ctgtactacc cgtctctgga
     1981 agaaatcgag aaagcgctga aagcgttcgc ggcgctgggt gttgaaatca tgatcaccga
     2041 actggacgtt aacacccagg gtgacgtttc tccggacgcg ctacaagaac aggcggaacg
     2101 tatgcgtgac ctgttcgaac tgttcaagaa acactctgac aaaatcaccg gtgttacctt
     2161 ctggggtgtt gcggacgacc agtcttggaa gaacaacttc ccggttccgg gtcgtaccaa
     2221 cgcgccgctg ctgttcgacc gtaactacca gccgaaaccg gcgttctggg cgatcgttta
     2281 actgcaggcg agctccgtcg acaagcttgc ggccgcactc gagcaccacc accaccacca
     2341 ccaccactaa ttgattaata cctaggctgc taaacaaagc ccgaaaggaa gctgagttgg
     2401 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     2461 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
     2521 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     2581 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2641 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2701 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     2761 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     2821 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     2881 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     2941 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     3001 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
     3061 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     3121 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     3181 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     3241 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     3301 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     3361 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     3421 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     3481 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     3541 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     3601 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     3661 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     3721 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     3781 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     3841 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     3901 agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     3961 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4021 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4081 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     4141 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     4201 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     4261 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     4321 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     4381 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     4441 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4501 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     4561 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     4621 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     4681 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     4741 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     4801 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     4861 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     4921 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     4981 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     5041 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     5101 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     5161 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     5221 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     5281 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     5341 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     5401 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     5461 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     5521 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     5581 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     5641 aggagcacga tcatgctagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     5701 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     5761 gcgttgtgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     5821 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     5881 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     5941 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     6001 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tgtccgcacc
     6061 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     6121 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     6181 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     6241 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     6301 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     6361 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     6421 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     6481 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     6541 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     6601 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     6661 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     6721 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     6781 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     6841 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     6901 gcgaaaggtt ttgcgccatt cgatggtgtc ctggatctcg acgctctccc ttatgcgact
     6961 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     7021 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     7081 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     7141 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     7201 gtccggcgta gaggatcgag atcgatctcg atcccgcgaa at
//
