LOCUS       sequence-206275-        7248 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Xylanase design 8.2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7248)
  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 7248)
  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: xyl8.2
FEATURES             Location/Qualifiers
     source          1..7248
                     /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             2330..2353
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2416..2434)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2430..2477
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2514..2969
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2601..2620)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2811..2832
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3061..3876)
                     /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(3061..3876)
                     /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     3784..3803
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3868..3887
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3998..4586
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4487..4506
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4740..4757
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4772..4911
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4837..4856)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4997..5019
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5013..5204)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5776..6858)
                     /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     6820..6839
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6859..6936)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7142..7161
                     /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 ggtgcggcgg ttgaaccgtc tcagctgtct ggtccgcagg cgaaactgat
     1381 ccagaaacac ttcaacatgc tggttgcggc gaacgcgatg aaaccggaat ctctacaacc
     1441 gaccgaaggt aacttcacct tcgacaacgc ggacaaaatc gttgagtttg cgatcgcgaa
     1501 caacatgaaa atgcgtggtc acaccctggt ttggcacaac caggttccgg actggttctt
     1561 ccaggacccg accgacccgt ctaaaccggc gacccgtgaa cagctgcgtg aacgtatgcg
     1621 tgaacacatc cgtaccgttc tggaccactt ccgtgacaaa ttcggttcta acaacccgat
     1681 catcggttgg gacgttgtta acgaaccgct gtctgacaac ggtacctacc gtccgtctcc
     1741 gtggtaccag atcatcggtc cggactacat cgcggacgcg ttccgttggg cgcacgcggc
     1801 ggacccgtct gcgaaactgt acctgaacga ctacggtatc gaaggtaacg gtgcgaaatc
     1861 tgacgcgatg tacaacctgg ttaaagacct acaagaccgt ggtgttccga tccacggtat
     1921 cggtatgcag ggtcacaccc acatcaactc taacccggaa tggatcgaac aggcgatcga
     1981 acgtttcgcg tctctgggtg ttgaagttca catcaccgaa ctggacgttt ctatgttcgc
     2041 gtgggacgac aaacgtaccg acgttaccga accgaccgaa gaaatgctgg aacgtcaggc
     2101 ggaacgttac cgtgaactgt tcgaaatctt ccgtaaacac tctgacgtta tcacctctgt
     2161 taccttctgg ggtgttgcgg acggtcactc ttggctgaac aacttcccgg ttaaaggtcg
     2221 taccgactac ccgctgctgt tcgaccgtaa ctaccagccg aagaaagcgt tctgggcgat
     2281 cgtttaactg caggcgagct ccgtcgacaa gcttgcggcc gcactcgagc accaccacca
     2341 ccaccaccac cactaattga ttaataccta ggctgctaaa caaagcccga aaggaagctg
     2401 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     2461 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
     2521 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2581 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2641 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2701 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2761 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2821 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2881 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2941 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     3001 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc
     3061 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     3121 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     3181 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     3241 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     3301 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
     3361 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     3421 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     3481 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     3541 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     3601 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     3661 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     3721 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     3781 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     3841 cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     3901 gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact
     3961 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4021 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4081 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4141 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4201 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4261 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4321 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4381 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4441 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4501 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4561 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4621 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4681 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     4741 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     4801 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     4861 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     4921 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     4981 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     5041 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     5101 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     5161 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     5221 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     5281 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     5341 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     5401 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     5461 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     5521 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     5581 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     5641 aacgacagga gcacgatcat gctagtcatg ccccgcgccc accggaagga gctgactggg
     5701 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     5761 ttaattgcgt tgtgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     5821 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     5881 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     5941 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     6001 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc
     6061 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     6121 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     6181 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     6241 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     6301 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     6361 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     6421 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     6481 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     6541 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     6601 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     6661 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     6721 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     6781 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     6841 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     6901 cataccgcga aaggttttgc gccattcgat ggtgtcctgg atctcgacgc tctcccttat
     6961 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     7021 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     7081 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     7141 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     7201 cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc cgcgaaat
//
