LOCUS       sequence-425190-        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  JOURNAL   Unpublished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            "Unpublished"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     5..24
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5..23
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    24..48
                     /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             63..85
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             108..137
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             156..311
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     CDS             1206..1223
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1276..1294)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1290..1337
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1374..1829
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1461..1480)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1671..1692
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1921..2736)
                     /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(1921..2736)
                     /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     2644..2663
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2728..2747
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2858..3446
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3347..3366
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3600..3617
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3632..3771
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3697..3716)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3857..3879
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3873..4064)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4873..5955)
                     /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     5917..5936
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5956..6033)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6239..6258
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
       61 attttgttta actttaagaa ggagatatac atatgtctaa agaaaaacac catcatcatc
      121 atcatcacca tcaccatgga tctgccaact caggcaccac gaatccaggc gtgtcagcct
      181 ggcaagtgaa caccgcctac acagcagggc agttggtcac gtataacggc aaaacgtata
      241 aatgccttca acctcacaca agtcttgccg gttgggagcc atccaacgta cccgccttat
      301 ggcaactgca atctggtaac ggatccgctg taaccgggac aacaaaggcg aacatcaagc
      361 ttttcagttt cactgaagtg aacgatacca acccgttaaa taacttgaat tttaccctga
      421 agaattcggg aaaacctctt gttgacatgg tcgttttgtt ctcggctaac atcaattatg
      481 acgctgctaa tgacaaggta ttcgtctcga acaaccctaa tgtgcagcat cttcttacga
      541 accgtgcaaa gtatttgaag ccattgcagg ataagggaat caaagtaatc ttatctattt
      601 tagggaatca tgatcgttcg gggatcgcga atctgtctac cgctcgcgcg aaagcctttg
      661 cgcaggaatt gaagaacact tgcgatttat ataacctgga cggcgtcttc tttgatgacg
      721 aatactccgc ataccagacc cctcccccga gtggttttgt caccccgagt aacaatgcgg
      781 ctgcacgtct ggcatacgag accaaacagg cgatgccgaa caagttggtt acagtatatg
      841 tgtattcacg cacaagctcc ttccctacag cggtcgacgg cgtaaatgcg gggtcctatg
      901 tcgattatgc aatccacgat tacggaggaa gttacgattt agctactaac tatcctggat
      961 tggcgaagag cggcatggtt atgtcatcgc aagagttcaa tcaaggccgt tacgcaaccg
     1021 ctcaggcgtt gcgcaacatt gttaccaaag gttacggagg tcacatgatt tttgcaatgg
     1081 accccaaccg cagcaacttc acttcagggc aattacctgc gttgaagctg attgctaagg
     1141 aactttatgg cgacgagctt gtctattcaa atacaccgta tagtaaggac tggtaatagc
     1201 tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg
     1261 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     1321 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
     1381 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     1441 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     1501 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     1561 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     1621 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     1681 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     1741 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     1801 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     1861 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc
     1921 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     1981 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     2041 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     2101 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     2161 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
     2221 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     2281 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     2341 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     2401 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     2461 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     2521 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     2581 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     2641 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     2701 cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     2761 gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact
     2821 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     2881 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     2941 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3001 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3061 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3121 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3181 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     3241 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     3301 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     3361 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     3421 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     3481 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     3541 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     3601 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     3661 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     3721 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     3781 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     3841 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     3901 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     3961 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     4021 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     4081 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     4141 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     4201 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     4261 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     4321 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     4381 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     4441 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     4501 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     4561 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     4621 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     4681 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     4741 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     4801 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     4861 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4921 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     4981 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     5041 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     5101 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatgtccgc
     5161 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     5221 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     5281 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     5341 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     5401 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     5461 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     5521 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     5581 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     5641 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     5701 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     5761 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     5821 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     5881 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     5941 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     6001 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     6061 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     6121 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     6181 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     6241 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     6301 tgcgtccggc gtagaggatc gagatcgatc tcgatcccgc g
//
