LOCUS       sequence-431796-        6341 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of a His-tagged Chitin Binding fusion of 
            Elizabethkingia miricola EndoF3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6341)
  TITLE     Deglycosylation Toolkit
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6341)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pET-29b(+)_His-CBD-EmEndoF3
FEATURES             Location/Qualifiers
     source          1..6341
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(158..1240)
                     /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     1202..1221
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1241..1318)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1524..1543
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1631..1650
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1631..1649
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1650..1674
                     /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             1689..1711
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1734..1763
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1782..1937
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     CDS             2832..2849
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2902..2920)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2916..2963
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3000..3455
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3087..3106)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3297..3318
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3547..4362)
                     /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(3547..4362)
                     /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     4270..4289
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4354..4373
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4484..5072
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4973..4992
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5226..5243
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5258..5397
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5323..5342)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5483..5505
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5499..5690)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
       61 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
      121 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
      181 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
      241 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
      301 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
      361 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
      421 cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
      481 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
      541 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
      601 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
      661 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
      721 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
      781 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
      841 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
      901 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
      961 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     1021 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     1081 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     1141 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     1201 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     1261 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     1321 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     1381 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     1441 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     1501 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct
     1561 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat cgatctcgat
     1621 cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta
     1681 gaaataattt tgtttaactt taagaaggag atatacatat gtctaaagaa aaacaccatc
     1741 atcatcatca tcaccatcac catggatctg ccaactcagg caccacgaat ccaggcgtgt
     1801 cagcctggca agtgaacacc gcctacacag cagggcagtt ggtcacgtat aacggcaaaa
     1861 cgtataaatg ccttcaacct cacacaagtc ttgccggttg ggagccatcc aacgtacccg
     1921 ccttatggca actgcaatct ggtaacggat ccactgcctt ggcaggctct aatggtgtct
     1981 gtatcgcgta ctacatcacc gacggacgta atcctacctt taagctgaaa gatatcccgg
     2041 ataaagtgga tatggttatt ctgttcggtc ttaaatattg gagtttgcaa gacacgacaa
     2101 agttgcctgg tggtacggga atgatgggta gcttcaaatc gtataaagac cttgatacgc
     2161 agattcgctc gcttcagtcc cgtgggatta aggttttgca aaatatcgac gacgacgttt
     2221 cttggcagag ctcaaaacct ggcggttttg cgagtgcagc tgcctacggc gatgccatta
     2281 aaagcattgt catcgacaaa tggaagttag acggaatcag tcttgatatc gaacactctg
     2341 gcgcaaaacc aaatcccatc ccaacttttc caggatatgc tgcgactggt tacaacggat
     2401 ggtattcagg gagtatggca gcgacccctg cttttctgaa tgtgatctcc gaattgacca
     2461 aatactttgg aacaaccgcg ccgaataaca agcaacttca gatcgcatcg ggtattgatg
     2521 tgtacgcctg gaacaagatt atggagaact tccgtaacaa cttcaattac atccagttac
     2581 agagttacgg ggcaaacgtg agccgtacgc agttgatgat gaattatgcg accggaacca
     2641 ataaaattcc ggcatctaag atggtttttg gggcctatgc agaaggaggc acgaatcagg
     2701 caaacgatgt cgaggtggct aaatggacgc ctactcaggg ggctaaaggt ggtatgatga
     2761 tttatactta caactccaac gtttcctatg ccaatgccgt tcgtgatgcg gtcaaaaact
     2821 aatagctcga gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg
     2881 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     2941 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga
     3001 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3061 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3121 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3181 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     3241 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3301 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     3361 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3421 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     3481 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat
     3541 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     3601 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     3661 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     3721 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     3781 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     3841 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     3901 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
     3961 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     4021 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     4081 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     4141 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     4201 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     4261 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     4321 tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
     4381 gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt
     4441 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     4501 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     4561 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     4621 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     4681 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     4741 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     4801 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4861 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     4921 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4981 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     5041 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     5101 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     5161 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     5221 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     5281 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     5341 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     5401 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     5461 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     5521 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     5581 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     5641 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     5701 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     5761 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     5821 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     5881 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     5941 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     6001 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     6061 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     6121 gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg
     6181 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     6241 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     6301 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc c
//
