LOCUS       sequence-375171-        6630 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses chymotrypsin inhibitor with an N-terminal MBP fusion 
            protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6630)
  AUTHORS   Raab SA, El-Baba TJ, Woodall DW, Liu W, Liu Y, Baird Z, Hales DA, 
            Laganowsky A, Russell DH, Clemmer DE
  TITLE     Evidence for Many Unique Solution Structures for Chymotrypsin 
            Inhibitor 2: A Thermodynamic Perspective Derived from vT-ESI-IMS-MS 
            Measurements.
  JOURNAL   J Am Chem Soc. 2020 Oct 14;142(41):17372-17383. doi: 
            10.1021/jacs.0c05365. Epub 2020 Sep 29.
  PUBMED    32866376
REFERENCE   2  (bases 1 to 6630)
  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"; doi: 
            "10.1021/jacs.0c05365"; journalName: "J Am Chem Soc"; date: 
            "2020-10-14- 14"; volume: "142"; issue: "41"; pages: "17372-17383"
COMMENT     Sequence Label: pET28-MBP-CI2F
FEATURES             Location/Qualifiers
     source          1..6630
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(487..564)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     770..789
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     873..892
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        873..891
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    892..916
                     /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             931..953
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             972..989
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             999..1016
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1020..2120
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     2094..2117
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2139..2159
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2367..2384
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2437..2455)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2451..2498
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2535..2990
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2622..2641)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2832..2853
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3082..3897)
                     /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(3082..3897)
                     /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     3805..3824
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3889..3908
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4019..4607
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4508..4527
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4761..4778
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4793..4932
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4858..4877)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5018..5040
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5034..5225)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6034..6630,1..486))
                     /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"
ORIGIN
        1 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
       61 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
      121 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
      181 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
      241 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
      301 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
      361 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
      421 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
      481 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
      541 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
      601 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
      661 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
      721 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
      781 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
      841 cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
      901 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca
      961 tgggcagcag ccatcatcat catcatcaca gcagcggcct ggtgccgcgc ggcagccata
     1021 tgaaaatcga agaaggtaaa ctggtaatct ggattaacgg cgataaaggc tataacggtc
     1081 tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat taaagtcacc gttgagcatc
     1141 cggataaact ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc cctgacatta
     1201 tcttctgggc acacgaccgc tttggtggct acgctcaatc tggcctgttg gctgaaatca
     1261 ccccggacaa agcgttccag gacaagctgt atccgtttac ctgggatgcc gtacgttaca
     1321 acggcaagct gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag
     1381 atctgctgcc gaacccgcca aaaacctggg aagagatccc ggcgctggat aaagaactga
     1441 aagcgaaagg taagagcgcg ctgatgttca acctgcaaga accgtacttc acctggccgc
     1501 tgattgctgc tgacgggggt tatgcgttca agtatgaaaa cggcaagtac gacattaaag
     1561 acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac cttcctggtt gacctgatta
     1621 aaaacaaaca catgaatgca gacaccgatt actccatcgc agaagctgcc tttaataaag
     1681 gcgaaacagc gatgaccatc aacggcccgt gggcatggtc caacatcgac accagcaaag
     1741 tgaattatgg tgtaacggta ctgccgacct tcaagggtca accatccaaa ccgttcgttg
     1801 gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa agagctggca aaagagttcc
     1861 tcgaaaacta tctgctgact gatgaaggtc tggaagcggt taataaagac aaaccgctgg
     1921 gtgccgtagc gctgaagtct tacgaggaag agttggcgaa agatccacgt attgccgcca
     1981 ctatggaaaa cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg tccgctttct
     2041 ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag
     2101 ccctgaaaga cgcgcagact aattcgagct cgcatatgga aaacctgtat tttcagggat
     2161 ccatgaagac ggaatggccg gagctggttg gcaaatctgt agaagaagcc aaaaaagtga
     2221 tcctgcaaga taaaccagag gctcaaatca ttgtactgcc ggtcggtacg attgtgacga
     2281 tggagtaccg tatcgaccgt gtgcgtttgt tcgtagataa attggacaac attgcgcaag
     2341 tcccgcgtgt tggttaatga ctcgagcacc accaccacca ccactgagat ccggctgcta
     2401 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     2461 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     2521 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2581 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2641 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2701 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     2761 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     2821 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     2881 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     2941 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     3001 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3061 tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     3121 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     3181 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     3241 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     3301 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt
     3361 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     3421 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg
     3481 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     3541 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     3601 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     3661 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     3721 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     3781 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     3841 catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac
     3901 accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt
     3961 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     4021 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4081 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4141 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     4201 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4261 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4321 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4381 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4441 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4501 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4561 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     4621 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     4681 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     4741 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     4801 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
     4861 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     4921 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     4981 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     5041 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     5101 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     5161 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     5221 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     5281 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     5341 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     5401 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     5461 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     5521 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     5581 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     5641 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc
     5701 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     5761 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     5821 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     5881 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     5941 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     6001 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     6061 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     6121 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     6181 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     6241 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     6301 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     6361 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     6421 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     6481 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     6541 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     6601 ctccacgccc agtcgcgtac cgtcttcatg
//
