LOCUS       sequence-431799-        6281 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of a His-tagged Chitin Binding fusion of 
            Streptomyces plicatus EndoH.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6281)
  TITLE     Deglycosylation Toolkit
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6281)
  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            "Unpublished"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-SpEndoH
FEATURES             Location/Qualifiers
     source          1..6281
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(234..1316)
                     /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     1278..1297
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1317..1394)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1600..1619
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1707..1726
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1707..1725
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1726..1750
                     /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             1765..1787
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1810..1839
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1858..2013
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     CDS             2848..2865
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2918..2936)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2932..2979
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3016..3471
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3103..3122)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3313..3334
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3563..4378)
                     /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(3563..4378)
                     /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     4286..4305
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4370..4389
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4500..5088
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4989..5008
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5242..5259
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5274..5413
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5339..5358)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5499..5521
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5515..5706)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
       61 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
      121 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
      181 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
      241 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
      301 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
      361 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
      421 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
      481 ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg caccaacgcg cagcccggac
      541 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
      601 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
      661 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
      721 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
      781 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
      841 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
      901 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
      961 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     1021 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     1081 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     1141 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     1201 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     1261 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     1321 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     1381 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     1441 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     1501 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     1561 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     1621 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     1681 cgagatcgat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     1741 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat acatatgtct
     1801 aaagaaaaac accatcatca tcatcatcac catcaccatg gatctgccaa ctcaggcacc
     1861 acgaatccag gcgtgtcagc ctggcaagtg aacaccgcct acacagcagg gcagttggtc
     1921 acgtataacg gcaaaacgta taaatgcctt caacctcaca caagtcttgc cggttgggag
     1981 ccatccaacg tacccgcctt atggcaactg caatctggta acggatccgc ccccgtaaaa
     2041 caagggccga cctccgtggc gtacgttgaa gtaaacaata atagcatgtt aaacgttggt
     2101 aaatacaccc ttgccgatgg cgggggtaat gcgttcgacg tggctgtcat ttttgctgca
     2161 aatatcaatt acgacaccgg gactaagaca gcttacttac atttcaacga gaacgtccaa
     2221 cgtgttttag acaacgccgt aacacaaatc cgtcctttgc aacaacaagg gatcaaagtg
     2281 ctgttgagcg ttttgggtaa ccatcagggg gctggatttg ctaattttcc ttcgcagcag
     2341 gccgcgtctg cgtttgcaaa acaactttca gatgcggtgg caaagtatgg ccttgatggg
     2401 gtggacttcg atgacgaata cgcagaatat ggcaataacg ggacagctca accgaacgat
     2461 agcagttttg tacacttggt cactgcgtta cgcgcaaaca tgccggacaa aatcattagc
     2521 ttgtacaaca ttggccctgc tgcgtcccgt ttgagctacg ggggagtcga tgtatcggat
     2581 aagtttgatt acgcttggaa tccatactat ggtacatggc aggttccagg tatcgcatta
     2641 ccaaaagctc agctttcgcc ggcggctgtt gaaatcggac gcacttcacg ctcaactgtc
     2701 gctgatctgg cacgccgtac tgtggacgaa ggttatggtg tatacctgac gtataacctt
     2761 gatggtggag accgtacagc cgatgtatcc gccttcacgc gtgagctgta tggctccgaa
     2821 gcagtacgta caccgtaata gctcgagcac caccaccacc accactgaga tccggctgct
     2881 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     2941 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     3001 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3061 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3121 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3181 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     3241 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3301 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     3361 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     3421 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     3481 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3541 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     3601 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     3661 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     3721 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     3781 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     3841 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     3901 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     3961 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     4021 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     4081 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     4141 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     4201 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     4261 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     4321 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     4381 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     4441 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     4501 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     4561 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     4621 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     4681 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     4741 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4801 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4861 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4921 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4981 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     5041 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     5101 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     5161 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     5221 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     5281 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     5341 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     5401 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     5461 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     5521 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     5581 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     5641 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     5701 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     5761 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     5821 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     5881 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     5941 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     6001 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     6061 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     6121 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     6181 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     6241 ggcttgagcg agggcgtgca agattccgaa taccgcaagc g
//
