LOCUS       sequence-206267-        7293 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Xylanase design 3.2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7293)
  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 7293)
  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: xyl3.2
FEATURES             Location/Qualifiers
     source          1..7293
                     /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             2375..2398
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2461..2479)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2475..2522
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2559..3014
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2646..2665)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2856..2877
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3106..3921)
                     /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(3106..3921)
                     /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     3829..3848
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3913..3932
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4043..4631
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4532..4551
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4785..4802
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4817..4956
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4882..4901)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5042..5064
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5058..5249)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5821..6903)
                     /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     6865..6884
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6904..6981)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7187..7206
                     /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 tccgaattca aaccgcacat ctctgcgctg aacgcgccgt ctctggcgga
     1321 acgttacaaa aactacttct acatcggtgc ggcggttgaa ccggaccaga ccaccaaaga
     1381 aaagaacgcg aaaatgctgc gtcgtcactt cgacatgatc gttgcggaaa acgcgatgaa
     1441 accggaagcg ctggaaccga ccgaaggtaa cttcaccttc gacaacgcga acaaaatcgt
     1501 tgacttcgcg atcgcgcaca acatgaaaat gcgtggtcac accctgctgt ggcacaacca
     1561 gatcccggaa tggttcttcc gtgacccgtc tgacccgtct aaaccggcgt ctcgtgacct
     1621 gctgttgcaa cgtctgcgtg accacatcac caccgttctg gaccacttca aaaccaaata
     1681 cggttctcag aacccgatca tcggttggga cgttgttaac gaagttctgg acgacaacgg
     1741 taacctgcgt aactctaaat tcttgcaaat catcggtccg gactacatcg aaaaagcgtt
     1801 ccgtttcgcg cacgaagcgg acccgtctgc gaaactgttc atgaacgact acaacaccga
     1861 acaggaaccg aaacgtcagg gtatctacga actgatcaaa aaactgaaag aacgtggtgc
     1921 gccgatcgac ggtgttggta tgcaggcgca cgttaccatc gactggccgc cggttgacga
     1981 aatcaagaaa gcgatccaga tgttcgcggc gctgggtctg gaagttcaga tcaccgaact
     2041 ggacgtttct ctgtacggtt ggccgccgcg tccggcgtac ccgacctacg acgcgatccc
     2101 ggaagaacgt ttccaggcgc aggcggaccg ttaccgtcag ctgttcgaac tgttcgaaga
     2161 actgaaagac aaaatctctg cggttacctt ctggggtatc gcggacaacc acacctggct
     2221 ggacgaccgt gcgcgtgaat acaacaacgg tgttggtaaa gacgcgccgt tcgttttcga
     2281 cccgaactac cgtgttaaac cggcgtactg ggcgatcatc aaccacaaat aactgcaggc
     2341 gagctccgtc gacaagcttg cggccgcact cgagcaccac caccaccacc accaccacta
     2401 attgattaat acctaggctg ctaaacaaag cccgaaagga agctgagttg gctgctgcca
     2461 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     2521 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
     2581 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     2641 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     2701 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     2761 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     2821 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     2881 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     2941 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     3001 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     3061 tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc
     3121 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     3181 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     3241 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     3301 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     3361 tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     3421 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     3481 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     3541 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     3601 gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     3661 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     3721 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     3781 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     3841 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt
     3901 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     3961 tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     4021 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     4081 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     4141 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     4201 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     4261 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     4321 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     4381 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     4441 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     4501 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     4561 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     4621 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     4681 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     4741 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     4801 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4861 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     4921 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     4981 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     5041 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     5101 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     5161 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     5221 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     5281 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     5341 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     5401 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     5461 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     5521 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     5581 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     5641 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     5701 atcatgctag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     5761 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgtgc
     5821 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5881 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     5941 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     6001 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     6061 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac caacgcgcag
     6121 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     6181 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     6241 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     6301 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     6361 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     6421 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     6481 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     6541 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     6601 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     6661 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     6721 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     6781 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     6841 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     6901 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     6961 tttgcgccat tcgatggtgt cctggatctc gacgctctcc cttatgcgac tcctgcatta
     7021 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     7081 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     7141 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     7201 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     7261 agaggatcga gatcgatctc gatcccgcga aat
//
