LOCUS       sequence-431793-        6335 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of a His-tagged Chitin Binding fusion of 
            Elizabethkingia miricola EndoF2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6335)
  TITLE     Deglycosylation Toolkit
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6335)
  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            "Unpublished"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(+)_notufA-EmEndoF2-CBD-His
FEATURES             Location/Qualifiers
     source          1..6335
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             658..813
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     CDS             826..855
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             868..885
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(938..956)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      952..999
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1036..1491
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1123..1142)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1333..1354
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1583..2398)
                     /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(1583..2398)
                     /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     2306..2325
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2390..2409
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2520..3108
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3009..3028
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3262..3279
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3294..3433
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3359..3378)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3519..3541
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3535..3726)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4535..5617)
                     /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     5579..5598
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5618..5695)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5901..5920
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6008..6027
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6008..6026
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6027..6051
                     /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             6066..6088
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 actaaggtca tcatcaccct tggagatttg aactcagcta cgacgacggg aggccaagac
       61 tcgatcggct atagttcttg ggctaaaggg atctatgaca aatgggttgg ggagtacaac
      121 ttagatggta ttgacatcga tattgaatct agcccatcag gagccacact tacgaaattt
      181 gtagctgcca cgaaggcgtt gtcaaagtat ttcggcccaa agtcggggac ggggaaaaca
      241 ttcgtgtacg acactaatca aaaccccacg aactttttta ttcagacggc tccgcgctac
      301 aattacgtgt ttttacaagc gtacggtcgc tctacaacca acttaactac ggtatctggc
      361 ttatacgcac cttacatttc catgaaacaa ttcctgcccg gatttagctt ctacgaagag
      421 aatggctacc ctgggaatta ttggaatgac gtgcgctatc cgcagaacgg gactggacgc
      481 gcttatgatt acgcacgctg gcaacctgcc actgggaaga agggtggcgt gtttagttac
      541 gctattgaac gtgacgctcc cttgacctcc agcaacgata acacattgcg tgctcccaac
      601 ttccgtgtta ctaaggattt aatcaagatt atgaatccgg gatccgccaa ctcaggcacc
      661 acgaatccag gcgtgtcagc ctggcaagtg aacaccgcct acacagcagg gcagttggtc
      721 acgtataacg gcaaaacgta taaatgcctt caacctcaca caagtcttgc cggttgggag
      781 ccatccaacg tacccgcctt atggcaactg caatctggta acggacacca tcatcatcat
      841 catcaccatc accattaata gctcgagcac caccaccacc accactgaga tccggctgct
      901 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
      961 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     1021 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     1081 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     1141 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     1201 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     1261 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     1321 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     1381 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     1441 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     1501 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     1561 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     1621 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     1681 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     1741 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     1801 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     1861 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     1921 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     1981 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     2041 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     2101 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     2161 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     2221 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     2281 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     2341 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     2401 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     2461 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     2521 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     2581 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     2641 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     2701 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     2761 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     2821 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     2881 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     2941 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     3001 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     3061 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     3121 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     3181 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     3241 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     3301 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     3361 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     3421 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     3481 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     3541 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     3601 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     3661 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     3721 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     3781 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     3841 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     3901 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     3961 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     4021 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     4081 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     4141 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     4201 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     4261 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     4321 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     4381 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     4441 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     4501 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4561 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4621 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     4681 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     4741 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     4801 atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     4861 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     4921 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     4981 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     5041 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     5101 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     5161 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     5221 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     5281 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     5341 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     5401 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     5461 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     5521 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     5581 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     5641 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     5701 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     5761 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     5821 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     5881 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     5941 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatcga tctcgatccc
     6001 gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa
     6061 ataattttgt ttaactttaa gaaggagata tacatatgaa atccgcggtg aacttatcca
     6121 atcttattgc ctataagaac agtgaccacc agatctccgc gggatactat cgcacatggc
     6181 gtgatagtgc gaccgcatcc ggcaaccttc ctagtatgcg ctggctgcca gattcacttg
     6241 atatggttat ggtttttcca gattacacac cgccggaaaa cgcctattgg aacaccttga
     6301 agacgaacta cgttccttat cttcacaaac gcgga
//
