LOCUS       sequence-206274-        7320 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Xylanase design 4.1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7320)
  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 7320)
  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: xyl4.1
FEATURES             Location/Qualifiers
     source          1..7320
                     /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             2402..2425
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2488..2506)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2502..2549
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2586..3041
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2673..2692)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2883..2904
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3133..3948)
                     /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(3133..3948)
                     /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     3856..3875
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3940..3959
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4070..4658
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4559..4578
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4812..4829
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4844..4983
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4909..4928)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5069..5091
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5085..5276)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5848..6930)
                     /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     6892..6911
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6931..7008)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7214..7233
                     /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 cccgtctctg tacgaagttt tcaaagacta
     1321 cttcccgatc ggtgttgcgg ttgcggcgac cggtggtaac gcggacctgt tcacctcttc
     1381 tgcggcgcag gacatggttc gtcagcactt caaccagatc accgcggaaa acgaaatgaa
     1441 accggaacac ctacaaccgg aagaaggtcg tttcaccttc gaagcggcgg accgtatggt
     1501 tgacttcgcg cagaaacacg gtatgaaagt tcgtggtcac accctggttt ggcacaacca
     1561 gaccccggac tggatgttca aagacgacaa cggtcagccg gtttctcgtg aacagctgct
     1621 ggaacgtatg cgtaaccaca tcaaaaccgt tgttggtcgt tacaaaggta aagtttactg
     1681 ctgggacgtt gttaacgaag cgatctctga caacggttct gactgcctgc gtgactctcg
     1741 ttggcgtcag atcatcggtg acgacttcat ggaagaagcg ttccgttacg cgcacgaagc
     1801 ggacccggac gcgaaactgt tcatgaacga ctacaacatc gaatctaacg gtgcgaaaac
     1861 cgacgcgatg tacaacctgg ttaaagagtt tctggcgcgt ggtgttccga tccacggtgt
     1921 tggtttccag ggtcacatcg gtctggactg gccgtctctg gacgaaatcg aagaagcgat
     1981 ccagcgtatc gcggacctgg gtctggacgt tgcgatcacc gaactggacg tttctatgta
     2041 cggttggccg ccgcgtgcgt acccgaccta cgacgcgatc ccggaacaga aattcctgga
     2101 ccaggcggaa cgttaccgtg aactgatgca ggcgttccgt aaatacgcgg accacatctc
     2161 tgcggttacc ttctggggta tcgcggacaa ccacacctgg ctggactctc gtgcggacgt
     2221 ttactacgac tctaacggta acgttgttac cgacccgaac gcgccgtacg cgcgtgttga
     2281 gaaaggtaaa ggtaaagacg cgccgttcgt tttcggtccg gactacaacg ttaaaccggc
     2341 gttctgggcg atcgtttaac tgcaggcgag ctccgtcgac aagcttgcgg ccgcactcga
     2401 gcaccaccac caccaccacc accactaatt gattaatacc taggctgcta aacaaagccc
     2461 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     2521 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     2581 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     2641 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     2701 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     2761 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     2821 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     2881 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     2941 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     3001 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     3061 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3121 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     3181 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     3241 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     3301 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     3361 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     3421 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     3481 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     3541 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     3601 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     3661 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     3721 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     3781 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     3841 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     3901 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt
     3961 attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt
     4021 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4081 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4141 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4201 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     4261 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     4321 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     4381 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     4441 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     4501 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     4561 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     4621 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     4681 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     4741 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     4801 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     4861 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     4921 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     4981 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     5041 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     5101 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     5161 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     5221 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     5281 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     5341 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     5401 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     5461 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     5521 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     5581 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     5641 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     5701 agccgggtcc tcaacgacag gagcacgatc atgctagtca tgccccgcgc ccaccggaag
     5761 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     5821 agctaactta cattaattgc gttgtgctca ctgcccgctt tccagtcggg aaacctgtcg
     5881 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5941 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     6001 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     6061 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     6121 taccgagatg tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     6181 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     6241 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     6301 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     6361 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     6421 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     6481 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     6541 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     6601 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     6661 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     6721 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     6781 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     6841 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     6901 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     6961 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtcct ggatctcgac
     7021 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     7081 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     7141 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     7201 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     7261 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat cgatctcgat cccgcgaaat
//
