LOCUS       sequence-206273-        7269 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Xylanase design 3.3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7269)
  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 7269)
  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: xyl3.3
FEATURES             Location/Qualifiers
     source          1..7269
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..21
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2..20
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    21..45
                     /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             60..82
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             102..119
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             120..1217
                     /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      1074..1083
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1242..1262
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2352..2375
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2438..2456)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2452..2499
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2536..2991
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2623..2642)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2833..2854
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3083..3898)
                     /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(3083..3898)
                     /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     3806..3825
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3890..3909
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4020..4608
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4509..4528
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4762..4779
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4794..4933
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4859..4878)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5019..5041
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5035..5226)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5798..6880)
                     /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     6842..6861
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6881..6958)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7164..7183
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt
       61 ttgtttaact ttaagaagga gatatacata tgggcagcag ccatcatcat catcatcaca
      121 aaatcgaaga aggtaaactg gtaatctgga ttaacggcga taaaggctat aacggtctcg
      181 ctgaagtcgg taagaaattc gagaaagata ccggaattaa agtcaccgtt gagcatccgg
      241 ataaactgga agagaaattc ccacaggttg cggcaactgg cgatggccct gacattatct
      301 tctgggcaca cgaccgcttt ggtggctacg ctcaatctgg cctgttggct gaaatcaccc
      361 cggacaaagc gttccaggac aagctgtatc cgtttacctg ggatgccgta cgttacaacg
      421 gcaagctgat tgcttacccg atcgctgttg aagcgttatc gctgatttat aacaaagatc
      481 tgctgccgaa cccgccaaaa acctgggaag agatcccggc gctggataaa gaactgaaag
      541 cgaaaggtaa gagcgcgctg atgttcaacc tgcaagaacc gtacttcacc tggccgctga
      601 ttgctgctga cgggggttat gcgttcaagt atgaaaacgg caagcacgac attaaagacg
      661 tgggcgtgga taacgctggc gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa
      721 acaaacacat gaatgcagac accgattact ccatcgcaga agctgccttt aataaaggcg
      781 aaacagcgat gaccatcaac ggcccgtggg catggtccaa catcgacacc agcaaagtga
      841 attatggtgt aacggtactg ccgaccttca agggtcaacc atccaaaccg ttcgttggcg
      901 tgctgagcgc aggtattaac gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg
      961 aaaactatct gctgactgat gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg
     1021 ccgtagcgct gaagtcttac gaggaagagt tggcgaaaga tccacgtatt gccgccacca
     1081 tggaaaacgc ccagaaaggt gaaatcatgc cgaacatccc gcagatgtcc gctttctggt
     1141 atgccgtgcg tactgcggtg atcaacgccg ccagcggtcg tcagactgtc gatgaagccc
     1201 tgaaagacgc gcagactact agtggttctg gttccgcggg tgaaaacctg tacttccagg
     1261 gtaccattgg atccgaattc aaaccgcaca tctctgcgct gaacgcgccg tctctggcgc
     1321 agcgtttcaa agactacttc cgtgttggtg cggcggttca cccgtctcag accaccaaag
     1381 agaaagacgc gaaaatgctg cgtcgtcact tcaacgcgat caccccggaa aactctatga
     1441 aatggggtgt tttgcaagac gcgcagggtc gttggaactg gcgtgacgcg gacgcgttcg
     1501 ttaacttcgg tgaaaaacac aacatgaaca tccgtggtca caccctggtt tggcacgcgc
     1561 agatcccgga ctgggttttc aaaaacccgg acggttctta catctctaaa gaagcgctgc
     1621 tgaaacgtat ggaaaaccac atcaccaccc tggttggtcg ttacaaaggt aaagttcacg
     1681 cgtgggacgt tgttaacgaa gcggttggtg aagacggtcg tatgcgtgac tctcactggt
     1741 atcgtatcgt tggtgacgac ttcatccgtg acgcgttccg ttacgcgcac gaagcggacc
     1801 cggacgcgaa actgttctac aacgactaca acaccgaagt tgaaggtaaa cgtgaagcga
     1861 tctaccgtct gatcgaaaaa ctgaagaaga aaggtgttcc gatcgacggt gttggtatcc
     1921 aggcgcacgt taccatcgac cgtccgccgg ttgacgaaat caagaaaacc atccagcgtt
     1981 tcgcggacct gggtctggaa gttcagatca ccgaactgga cgtttctctg tacggttggc
     2041 cgccgcgtcc ggcgtacccg acctacgacg cgatcccgga agaacgtttc caggcgcagg
     2101 cggaccgtta ccgtcagctg ttcgaactgt tcgaagaaat gaaagacaaa ctgtcttctg
     2161 ttaccttctg gggtatcgcg gacaaccaca cctggctgga cgaccgtgcg cgtgaataca
     2221 acaacggtgt tggtaaagac gcgccgttcg ttttcgaccc gaactaccgt gttaaaccgg
     2281 cgtactgggc gatcatcaac cacaaataac tgcaggcgag ctccgtcgac aagcttgcgg
     2341 ccgcactcga gcaccaccac caccaccacc accactaatt gattaatacc taggctgcta
     2401 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     2461 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     2521 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2581 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2641 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2701 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     2761 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     2821 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     2881 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     2941 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     3001 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3061 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     3121 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     3181 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     3241 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     3301 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     3361 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     3421 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     3481 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     3541 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     3601 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     3661 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     3721 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     3781 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     3841 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     3901 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     3961 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     4021 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     4081 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     4141 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     4201 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     4261 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4321 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4381 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4441 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4501 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4561 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4621 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     4681 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     4741 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     4801 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     4861 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     4921 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     4981 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     5041 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     5101 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     5161 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     5221 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     5281 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     5341 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     5401 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     5461 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     5521 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     5581 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     5641 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgctagtca tgccccgcgc
     5701 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     5761 ctaatgagtg agctaactta cattaattgc gttgtgctca ctgcccgctt tccagtcggg
     5821 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5881 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     5941 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     6001 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     6061 cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     6121 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     6181 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     6241 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     6301 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     6361 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     6421 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     6481 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     6541 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     6601 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     6661 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     6721 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     6781 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     6841 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     6901 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtcct
     6961 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     7021 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     7081 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     7141 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct
     7201 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat cgatctcgat
     7261 cccgcgaaa
//
