LOCUS       sequence-206270-        7311 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Xylanase design 3.1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7311)
  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 7311)
  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: xyl3.1
FEATURES             Location/Qualifiers
     source          1..7311
                     /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             2393..2416
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2479..2497)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2493..2540
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2577..3032
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2664..2683)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2874..2895
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3124..3939)
                     /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(3124..3939)
                     /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     3847..3866
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3931..3950
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4061..4649
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4550..4569
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4803..4820
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4835..4974
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4900..4919)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5060..5082
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5076..5267)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5839..6921)
                     /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     6883..6902
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6922..6999)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7205..7224
                     /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 ctctggcgca
     1321 gcgttacaaa gactacttct acatcggtgc ggcggttgaa ccgtaccaga ccaccaaaga
     1381 aaaagacgcg aaaatgttgc aacgtcactt caacatgatc gttgcggaaa acgcgatgaa
     1441 accggcggcg ctggaaccga ccgaaggtaa cttccagtgg gcggacgcgg accgtatcgt
     1501 tcagttcgcg aaagaaaacg gtatggaact gcgtttccac accctggttt ggcacaacca
     1561 gaccccggac tggttcttcc tggaccgtga aggtaaaccg atggttgaag aaaccgaccc
     1621 gcagaaacgt gaagaaaacc gtgaactgct gttgcaacgt ctggaaaacc acatccgtgc
     1681 ggttgttctg cgttacaaag acgacatcaa aaactgggac gttgttaacg aagttgttga
     1741 accgaacgac ccgggtggta tgcgtaactc tccgtggtat cagatcaccg gtactgaata
     1801 catcgaagtt gcgttccgtg cggcgcgtga agcgggtggt gaagacatca aactgtacat
     1861 gaacgactac aacaccgaac aggaaccgaa acgtgaatac atctaccgtc tgatcaagaa
     1921 actgctggaa aaaggtgttc cgatcgacgg tgttggtcac caggcgcacg ttaccatcga
     1981 ccgtccgccg gttgacgaaa tcaagaaaac catccagcgt ttcgcggacc tgggtctgga
     2041 caaccagatc accgaactgg acgtttctct gtacggttgg ccgccgcgtc cggcgtaccc
     2101 gacctacgac gcgatcccgg aagaacgttt ccaggcgcag gcggaccgtt accgtcagct
     2161 gttcgaactg ttcgaagaac tgaaagacca catctctgcg gttaccttct ggggtatcgc
     2221 ggacaaccac acctggctgg acgaccgtgc gcgtgaatac aacgacggtg ttggtaaaga
     2281 cgcgccgttc gttttcgacc cgaactaccg tgttaaaccg gcgtactggg cgatcatcaa
     2341 ccacaaataa ctgcaggcga gctccgtcga caagcttgcg gccgcactcg agcaccacca
     2401 ccaccaccac caccactaat tgattaatac ctaggctgct aaacaaagcc cgaaaggaag
     2461 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     2521 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     2581 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     2641 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     2701 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     2761 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     2821 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     2881 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     2941 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     3001 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     3061 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa
     3121 ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
     3181 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
     3241 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
     3301 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
     3361 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg
     3421 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
     3481 ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat
     3541 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     3601 atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc
     3661 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     3721 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     3781 aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc
     3841 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     3901 cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt
     3961 tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc
     4021 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     4081 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     4141 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4201 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4261 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4321 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4381 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4441 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4501 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4561 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4621 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4681 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     4741 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     4801 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     4861 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     4921 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     4981 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     5041 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     5101 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     5161 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     5221 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     5281 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     5341 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     5401 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     5461 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     5521 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     5581 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     5641 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     5701 ctcaacgaca ggagcacgat catgctagtc atgccccgcg cccaccggaa ggagctgact
     5761 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     5821 acattaattg cgttgtgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5881 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     5941 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     6001 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     6061 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     6121 gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     6181 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     6241 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     6301 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     6361 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     6421 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     6481 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     6541 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     6601 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     6661 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     6721 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     6781 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     6841 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     6901 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     6961 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc tggatctcga cgctctccct
     7021 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     7081 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     7141 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     7201 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     7261 ccacgatgcg tccggcgtag aggatcgaga tcgatctcga tcccgcgaaa t
//
