LOCUS       sequence-343510-        7439 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses B. licheniformis chitinase with an N terminal 10X His tag 
            and HRV-3C cleavage site in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7439)
  AUTHORS   Bartholomai BM, Gladfelter AS, Loros JJ, Dunlap JC
  TITLE     Quantitative single molecule RNA-FISH and RNase-free cell wall 
            digestion in Neurospora crassa.
  JOURNAL   Fungal Genet Biol. 2021 Aug 20;156:103615. doi: 
            10.1016/j.fgb.2021.103615.
  PUBMED    34425213
REFERENCE   2  (bases 1 to 7439)
  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: "Fungal 
            Genet Biol."; date: "2021-08-20"; volume: "156:103615. doi"; pages: 
            " 10.1016/j.fgb.2021.103615"
COMMENT     Sequence Label: pET16better_H10_HRV3C_chitinase
FEATURES             Location/Qualifiers
     source          1..7439
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..139
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(140..208,209..1000)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(358..377)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1171..1759
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1660..1679
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1913..1930
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1945..2084
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2010..2029)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2170..2192
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2186..2377)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3689..4771)
                     /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     4733..4752
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4772..4849)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5055..5074
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5158..5177
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5158..5176
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5177..5201
                     /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             5216..5238
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5251..5280
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5293..5316
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     terminator      7111..7158
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(7333..7338,7339..7355,7356..7361))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(7388..7406)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
       61 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
      121 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
      181 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
      241 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
      301 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
      361 atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca
      421 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
      481 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
      541 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
      601 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
      661 cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg
      721 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
      781 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
      841 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
      901 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
      961 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     1021 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     1081 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     1141 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     1201 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     1261 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     1321 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     1381 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     1441 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     1501 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     1561 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     1621 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     1681 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     1741 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     1801 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     1861 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     1921 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     1981 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     2041 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     2101 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     2161 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     2221 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     2281 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     2341 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     2401 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     2461 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     2521 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     2581 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     2641 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     2701 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     2761 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     2821 ggagcacgat catgcgcacc cgtggccagg acccaacgct gcccgagatg cgccgcgtgc
     2881 ggctgctgga gatggcggac gcgatggata tgttctgcca agggttggtt tgcgcattca
     2941 cagttctccg caagaattga ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt
     3001 gccgccggct tccattcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg
     3061 gaggcagaca aggtataggg cggcgcctac aatccatgcc aacccgttcc atgtgctcgc
     3121 cgaggcggca taaatcgccg tgacgatcag cggtccaatg atcgaagtta ggctggtaag
     3181 agccgcgagc gatccttgaa gctgtccctg atggtcgtca tctacctgcc tggacagcat
     3241 ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa tggggaaggc
     3301 catccagcct cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc
     3361 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     3421 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     3481 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     3541 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     3601 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     3661 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     3721 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3781 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     3841 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     3901 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     3961 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     4021 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     4081 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     4141 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     4201 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     4261 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     4321 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     4381 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     4441 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     4501 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     4561 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     4621 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     4681 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     4741 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     4801 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     4861 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     4921 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     4981 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     5041 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     5101 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa
     5161 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt
     5221 ttaactttaa gaaggagata taccatgggc catcatcatc atcatcatca tcatcatcac
     5281 agcagcggcc atctggaagt gctgtttcag ggcccgaaaa tcgtgttgat caacaaaagc
     5341 aaaaagtttt tcgttttttc tttcattttt gttatgatgc tgagcctctc atttgtgaat
     5401 ggggaagttg caaaagccga ttccggaaaa aactataaaa tcatcggcta ctatccatca
     5461 tggggtgctt atggaaggga ttttcaagtt tgggatatgg acgtttcgaa agtcagccac
     5521 attaattatg cctttgctga tatttgctgg gagggaaggc atggaaaccc tgatccgaca
     5581 ggccccaatc ctcaaacgtg gtcatgccag gatgaaaacg gagtgatcga cgcgccaaat
     5641 ggaacaatcg tgatgggcga tccctggatt gacgcacaaa agtcaaatcc cggggatgtc
     5701 tgggatgaac cgatccgcgg caactttaaa caattgttga agctgaaaaa gagccaccct
     5761 catttgaaaa cgttcatatc ggtcgggggg tggacttggt ctaaccgctt ttcagatgtc
     5821 gcggcagatc ctgcggcaag ggagaatttc gccgcttcgg ccgttgagtt tttaaggaaa
     5881 tacgggtttg acggggtcga tcttgactgg gaatatccgg tcagcggagg attgccgggg
     5941 aacagcacac gtccggaaga taaaagaaac tacacgctgc tcctgcaaga ggtgcgcaaa
     6001 aaacttgacg ctgcagaagc aaaagacggc aaggaatact tgctgacgat cgcatccggc
     6061 gcaagtcccg attatgtaag caacactgag ctcgataaaa tcgctcaaac cgtggattgg
     6121 attaacatta tgacctatga ctttaatggc ggatggcaaa gcataagcgc ccataatgca
     6181 ccgctgttct atgatccaaa agcgaaagaa gcaggcgttc caaacgctga gacctacaat
     6241 attgaaaaca ctgtgaaacg ctacaaggaa gccggtgtca agggtgacaa attagtgctt
     6301 ggaacaccgt tctacggaag gggctggagc ggttgtgaac caggggggca cggagaatat
     6361 cagaaatgcg gaccggctaa agaagggaca tgggaaaagg gcgtattcga tttttcagat
     6421 cttgaaagga actatgtgaa tcaaaacggc tataaaaggt attggaacga tcaagcaaaa
     6481 gtgccgtttt tgtataatgc ggaaaatggc aatttcatca cttatgatga tgaacaatca
     6541 ttcggccaca aaacggattt tattaaagca aacggattaa gcggagcaat gttctgggat
     6601 ttcagcggcg attccaatcg gacgcttctc aataaattgg cagccgattt agattttgca
     6661 ccggacggag gcaatccgga gccgccttca tcggcacctg tgaatgtgcg tgtaaccgga
     6721 aaaactgcta caagtgtcag cctggcgtgg gatgcgccga gcagcggagc aaacattgcg
     6781 gaatatgtcg tgtcatttga aaaccggtcg atatctgtaa aagaaacatc agcggaaata
     6841 ggcggcttga agccgggtac ggcctactca tttactgttt cagcaaagga tgcggatgga
     6901 aagctccatg ccggaccaac ggtagaggtc acgacgaatt ctgaccaagc ctgttcatat
     6961 gacgaatgga aagagacgag cgcatacaca ggcggagagc gggttgcatt taacggaaaa
     7021 gtgtatgaag cgaaatggtg gacgaaaggc gaccggcctg atcaatccgg tgaatggggc
     7081 gtatggcggc tgatcggagg ctgcgaataa ctagcataac cccttggggc ctctaaacgg
     7141 gtcttgaggg gttttttgct gaaaggagga actatatccg gatatcccgc aagaggcccg
     7201 gcagtaccgg cataaccaag cctatgccta cagcatccag ggtgacggtg ccgaggatga
     7261 cgatgagcgc attgttagat ttcatacacg gtgcctgact gcgttagcaa tttaactgtg
     7321 ataaactacc gcattaaagc ttatcgatga taagctgtca aacatgagaa ttcttgaaga
     7381 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttc
//
