LOCUS       sequence-431792-        6341 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of a His-tagged Chitin Binding fusion of 
            Elizabethkingia miricola EndoF1.
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  JOURNAL   Unpublished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            "Unpublished"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-EmEndoF1
FEATURES             Location/Qualifiers
     source          1..6341
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     161..180
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     268..287
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        268..286
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    287..311
                     /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             326..348
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             371..400
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             419..574
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     CDS             1469..1486
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1539..1557)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1553..1600
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1637..2092
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1724..1743)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1934..1955
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2184..2999)
                     /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(2184..2999)
                     /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     2907..2926
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2991..3010
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3121..3709
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3610..3629
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3863..3880
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3895..4034
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3960..3979)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4120..4142
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4136..4327)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5136..6218)
                     /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     6180..6199
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6219..6296)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
       61 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
      121 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
      181 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
      241 tcgagatcga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
      301 ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata tacatatgtc
      361 taaagaaaaa caccatcatc atcatcatca ccatcaccat ggatctgcca actcaggcac
      421 cacgaatcca ggcgtgtcag cctggcaagt gaacaccgcc tacacagcag ggcagttggt
      481 cacgtataac ggcaaaacgt ataaatgcct tcaacctcac acaagtcttg ccggttggga
      541 gccatccaac gtacccgcct tatggcaact gcaatctggt aacggatccg ctgtaaccgg
      601 gacaacaaag gcgaacatca agcttttcag tttcactgaa gtgaacgata ccaacccgtt
      661 aaataacttg aattttaccc tgaagaattc gggaaaacct cttgttgaca tggtcgtttt
      721 gttctcggct aacatcaatt atgacgctgc taatgacaag gtattcgtct cgaacaaccc
      781 taatgtgcag catcttctta cgaaccgtgc aaagtatttg aagccattgc aggataaggg
      841 aatcaaagta atcttatcta ttttagggaa tcatgatcgt tcggggatcg cgaatctgtc
      901 taccgctcgc gcgaaagcct ttgcgcagga attgaagaac acttgcgatt tatataacct
      961 ggacggcgtc ttctttgatg acgaatactc cgcataccag acccctcccc cgagtggttt
     1021 tgtcaccccg agtaacaatg cggctgcacg tctggcatac gagaccaaac aggcgatgcc
     1081 gaacaagttg gttacagtat atgtgtattc acgcacaagc tccttcccta cagcggtcga
     1141 cggcgtaaat gcggggtcct atgtcgatta tgcaatccac gattacggag gaagttacga
     1201 tttagctact aactatcctg gattggcgaa gagcggcatg gttatgtcat cgcaagagtt
     1261 caatcaaggc cgttacgcaa ccgctcaggc gttgcgcaac attgttacca aaggttacgg
     1321 aggtcacatg atttttgcaa tggaccccaa ccgcagcaac ttcacttcag ggcaattacc
     1381 tgcgttgaag ctgattgcta aggaacttta tggcgacgag cttgtctatt caaatacacc
     1441 gtatagtaag gactggtaat agctcgagca ccaccaccac caccactgag atccggctgc
     1501 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
     1561 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
     1621 cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     1681 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     1741 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     1801 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     1861 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     1921 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     1981 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     2041 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     2101 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     2161 tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat
     2221 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     2281 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     2341 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     2401 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
     2461 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     2521 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     2581 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     2641 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     2701 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     2761 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     2821 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     2881 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     2941 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata
     3001 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc
     3061 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3121 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3181 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     3241 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     3301 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     3361 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     3421 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     3481 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3541 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3601 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3661 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3721 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     3781 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     3841 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     3901 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     3961 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     4021 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     4081 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     4141 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     4201 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     4261 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     4321 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     4381 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     4441 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     4501 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     4561 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     4621 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     4681 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     4741 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     4801 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     4861 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     4921 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     4981 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     5041 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     5101 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5161 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5221 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     5281 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     5341 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     5401 tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     5461 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     5521 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     5581 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     5641 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     5701 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     5761 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     5821 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     5881 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     5941 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     6001 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     6061 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     6121 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     6181 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     6241 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     6301 atctcgacgc tctcccttat gcgactcctg cattaggaag c
//
