LOCUS       sequence-206281-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Xylanase design 4.2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7302)
  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 7302)
  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: xyl4.2
FEATURES             Location/Qualifiers
     source          1..7302
                     /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             2384..2407
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2470..2488)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2484..2531
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2568..3023
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2655..2674)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2865..2886
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3115..3930)
                     /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(3115..3930)
                     /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     3838..3857
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3922..3941
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4052..4640
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4541..4560
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4794..4811
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4826..4965
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4891..4910)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5051..5073
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5067..5258)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5830..6912)
                     /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     6874..6893
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6913..6990)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7196..7215
                     /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 agaccgacat cccggacctg tacgaagttt tcaaagacta
     1321 cttcccgatc ggtgcggcgg ttgaaccgcg tcagctgaac ggtccggaag gtaaactgat
     1381 caagaaacac ttcaactctc tgaccgcgga aaacgaaatg aatccggaac gtctggaacc
     1441 gcgtgaaggt gttttcaact tcgaaaacgc ggaccgtatc gttgacttcg cgcagaagaa
     1501 caacatgaaa gttcgtggtc acaccctggt ttggcacaac cagaccccgg actgggtttt
     1561 caaagacgac aacggtcagc cggtttctaa agaagaactg ctggaacgta tgcgtaacca
     1621 catcaaaacc gttgttggtc gttacaaagg taaaatctac gcgtgggacg ttgttaacga
     1681 agcggttgcg gacgaaggta ccgaagttct gcgtccgtct aaatggcgtg aaatcatcgg
     1741 tgaagactac atcgaagaag cgttccgtta cgcgcacgaa gcggacccgg acgcgaaact
     1801 gttcatgaac gactacaaca tcgaaggtat caacgcgaaa tctgacgcgc tgtacaacct
     1861 ggttaaacgt ctgaaagaac gtggtgttcc ggttgacggt atcggtttcc agatgcacat
     1921 caacatcaac ggtgttgaca tggacaactt ccgtaaagcg atcgaacgtt tcgcgtctct
     1981 gggtctggaa gttgcgatca ccgaactgga cgtttctatg tacggttggc cgccgcgtgc
     2041 gtacccgacc tacgacgcga tcccggaaca gaaattcctg gaccaggcgg aacgttacaa
     2101 agaactgatg aacgcgttcc gtgaatacgc ggaccacatc acctctgtta ccttctgggg
     2161 tatcgcggac aaccacacct ggctggactc tcgtgcggac gtttactacg actctaacgg
     2221 taacgttgtt accgacccga acgcgccgta cgcgcgtgtt gagaaaggta aaggtaaaga
     2281 cgcgccgttc gttttcggtc cggactacaa cgttaaaccg gcgtactggg cggttgttta
     2341 actgcaggcg agctccgtcg acaagcttgc ggccgcactc gagcaccacc accaccacca
     2401 ccaccactaa ttgattaata cctaggctgc taaacaaagc ccgaaaggaa gctgagttgg
     2461 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     2521 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
     2581 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     2641 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2701 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2761 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     2821 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     2881 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     2941 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     3001 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     3061 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
     3121 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     3181 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     3241 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     3301 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     3361 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     3421 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     3481 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     3541 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     3601 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     3661 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     3721 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     3781 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     3841 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     3901 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     3961 agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4021 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4081 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4141 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     4201 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     4261 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     4321 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     4381 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     4441 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     4501 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4561 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     4621 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     4681 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     4741 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     4801 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     4861 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     4921 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     4981 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     5041 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     5101 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     5161 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     5221 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     5281 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     5341 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     5401 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     5461 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     5521 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     5581 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     5641 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     5701 aggagcacga tcatgctagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     5761 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     5821 gcgttgtgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     5881 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     5941 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     6001 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     6061 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tgtccgcacc
     6121 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     6181 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     6241 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     6301 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     6361 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     6421 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     6481 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     6541 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     6601 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     6661 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     6721 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     6781 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     6841 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     6901 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     6961 gcgaaaggtt ttgcgccatt cgatggtgtc ctggatctcg acgctctccc ttatgcgact
     7021 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     7081 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     7141 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     7201 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     7261 gtccggcgta gaggatcgag atcgatctcg atcccgcgaa at
//
