LOCUS       sequence-431798-        6461 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of a His-tagged Chitin Binding fusion of 
            Elizabethkingia miricola PNGaseF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6461)
  TITLE     Deglycosylation Toolkit
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6461)
  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            "Unpublished"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-3C-EmPNGaseF
FEATURES             Location/Qualifiers
     source          1..6461
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(24..41)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1002..1025)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(1056..1211)
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     CDS             complement(1230..1259)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1282..1304
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1319..1343
                     /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)."
     primer_bind     complement(1343..1362)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1344..1362)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1450..1469)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1675..1752
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1753..2835
                     /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     complement(1772..1791)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3644..3835
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3829..3851)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3937..4076
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3992..4011
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4091..4108)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4262..4850)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4342..4361)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4961..4980)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4972..5787
                     /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             4972..5787
                     /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     complement(5045..5064)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5879..6334)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6016..6037)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6228..6247
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(6371..6418)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     6414..6432
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
ORIGIN
        1 gctttgttag cagccggatc tcagtggtgg tggtggtggt gctcgagcta ttaattcgta
       61 actacaggtg ccgagatggg agtattagat tttgcgatta caaatgatga aattgcgtag
      121 aaggcgtcgc cattggtccc gttgttcgtc cacgattgga acttgtactc atacgagaaa
      181 gtagatccag tcagggagtt attcagaacg tcaatacgcg taggaactgc cattccagga
      241 caccaacctg cgcgatctgg cgcccaattc ccaggtgatt ggttgttgat aggattggca
      301 ctacacccta aagctcctaa ctgatgttgg aacgtgttag cgttgttgat ggcaattgta
      361 tgggtgcgaa aacaccactc cgcacaacca cgcgagccag catcgtatgg tttagcatgg
      421 ccccagccgc tgatagtggt gcgcaaatat gccttctcgg tgtttgttgg caattggatg
      481 ttcttcttca gccctaatgt atgcgcttta ccatacggga ccccgtcgat ggaagatttg
      541 ttgtattgaa tgacggggac cacggcactg tacttatagt ccggggtgcc atacacgatg
      601 tcaaaatcga cactatattc gcggcccttg gccaaccagg tctcggtgta gattttaagc
      661 tctgtatttc cactaagtaa tgacttaaag tccgtaacat caatctccag tccacggggc
      721 aatttctcgg tgccgaccca gtaaggtgta atgaaacgtc cgatttcata ccattcaccg
      781 gtggtcttgt tcttgacata tacattagca tagcgatccc attcgtcgca tgttttatta
      841 gggcactcat ttttgatgaa cattttaata gttttgaccg ttgttacgtc cgccggaaag
      901 gtgaacgtcc cttcggcact ttggctcaac ccgtctccga aagcattctt gactttgtcg
      961 aacgtcttaa tattgaccgt gttatcggcc ggcgcggatc ctggaccttg gaacaatacc
     1021 tccaatccgg tagtcggaat atcgtagtca ccagattgca gttgccataa ggcgggtacg
     1081 ttggatggct cccaaccggc aagacttgtg tgaggttgaa ggcatttata cgttttgccg
     1141 ttatacgtga ccaactgccc tgctgtgtag gcggtgttca cttgccaggc tgacacgcct
     1201 ggattcgtgg tgcctgagtt ggcagatcca tggtgatggt gatgatgatg atgatggtgt
     1261 ttttctttag acatatgtat atctccttct taaagttaaa caaaattatt tctagagggg
     1321 aattgttatc cgctcacaat tcccctatag tgagtcgtat taatttcgcg ggatcgagat
     1381 cgatctcgat cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg
     1441 ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc
     1501 tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg
     1561 gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac
     1621 tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgagat cccggacacc
     1681 atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc
     1741 agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
     1801 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
     1861 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
     1921 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
     1981 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
     2041 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
     2101 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
     2161 gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc
     2221 aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca
     2281 ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg
     2341 cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg
     2401 gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc
     2461 atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc
     2521 attaccgagt ccgggctgcg cgttggtgcg gacatctcgg tagtgggata cgacgatacc
     2581 gaagacagct catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg
     2641 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat
     2701 cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc
     2761 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     2821 gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcaccggg
     2881 atctcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg
     2941 catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt
     3001 gccggcagcg ctctgggtca ttttcggcga ggaccgcttt cgctggagcg cgacgatgat
     3061 cggcctgtcg cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg
     3121 tcccgccacc aaacgtttcg gcgagaagca ggccattatc gccggcatgg cggccccacg
     3181 ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac
     3241 tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac
     3301 gtctgcgacc tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga
     3361 aacgcggaag tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg
     3421 gctaccctgt ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat
     3481 ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa
     3541 ccgggcatgt tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat
     3601 cattaccccc atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa
     3661 aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact
     3721 caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga
     3781 tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     3841 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     3901 tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag
     3961 cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg
     4021 caccattgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct
     4081 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4141 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4201 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4261 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4321 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4381 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4441 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4501 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4561 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4621 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4681 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     4741 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4801 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4861 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4921 tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat
     4981 attcaacggg aaacgtcttg ctctaggccg cgattaaatt ccaacatgga tgctgattta
     5041 tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg
     5101 tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat
     5161 gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc
     5221 atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggg
     5281 aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
     5341 ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc
     5401 gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg
     5461 agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat
     5521 aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac
     5581 cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
     5641 gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta
     5701 cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt
     5761 catttgatgc tcgatgagtt tttctaagaa ttaattcatg agcggataca tatttgaatg
     5821 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa
     5881 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     5941 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     6001 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     6061 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     6121 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     6181 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     6241 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     6301 cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccaatccg gatatagttc
     6361 ctcctttcag caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt tatgctagtt
     6421 attgctcagc ggtggcagca gccaactcag cttcctttcg g
//
