LOCUS       sequence-9746-ex        7463 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7463)
  AUTHORS   Hilf RJ, Dutzler R
  TITLE     Structure of a potentially open state of a proton-activated 
            pentameric ligand-gated ion channel.
  JOURNAL   Nature. 2009 Jan 1. 457(7225):115-8.
  PUBMED    18987630
REFERENCE   2  (bases 1 to 7463)
  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: "Nature. 
            2009 Jan 1. 457(7225):115-8."
COMMENT     Sequence Label: pET26-MBP-GLIC
FEATURES             Location/Qualifiers
     source          1..7463
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      13..468
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(100..119)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     310..331
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(561..1376)
                     /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(561..1376)
                     /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     1284..1303
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1368..1387
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1498..2086
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1987..2006
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2240..2257
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2272..2414
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2340..2359)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2500..2522
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2516..2707)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3516..4598)
                     /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     4560..4579
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4599..4676)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4882..4901
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4985..5004
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4985..5003
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5004..5028
                     /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             5043..5065
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     sig_peptide     5073..5138
                     /codon_start=1
                     /gene="pelB (fragment)"
                     /product="leader peptide for secretion"
                     /label=pelB signal sequence
                     /translation="MKYLLPTAAAGLLLLAAQPAMA"
     CDS             5178..5207
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5211..6311
                     /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     6285..6308
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             6321..6344
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             7308..7325
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7378..7396)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7392..7439
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
       61 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
      121 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
      181 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
      241 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
      301 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
      361 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
      421 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttca ggtggcactt
      481 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
      541 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
      601 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
      661 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
      721 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
      781 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
      841 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
      901 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
      961 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     1021 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     1081 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     1141 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     1201 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     1261 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     1321 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca
     1381 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta
     1441 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     1501 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     1561 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     1621 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     1681 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     1741 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     1801 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     1861 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     1921 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     1981 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     2041 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     2101 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     2161 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     2221 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     2281 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatatggtg cactctcagt
     2341 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     2401 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     2461 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     2521 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     2581 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     2641 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     2701 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     2761 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     2821 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     2881 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     2941 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     3001 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     3061 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     3121 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     3181 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     3241 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     3301 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     3361 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     3421 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     3481 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3541 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     3601 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     3661 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     3721 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     3781 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     3841 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     3901 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     3961 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     4021 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     4081 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     4141 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     4201 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     4261 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     4321 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     4381 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     4441 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     4501 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     4561 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     4621 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     4681 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     4741 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     4801 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     4861 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     4921 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     4981 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     5041 attttgttta actttaagaa ggagatatac atatgaaata cctgctgccg accgctgctg
     5101 ctggtctgct gctcctcgct gcccagccgg cgatggccat ggatatcgga attaattcgg
     5161 atccgaattc gagctcacat caccatcacc atcaccatca ccatcacccc atgaaaatcg
     5221 aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt ctcgctgaag
     5281 tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat ccggataaac
     5341 tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt atcttctggg
     5401 cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc accccggaca
     5461 aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac aacggcaagc
     5521 tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa gatctgctgc
     5581 cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg aaagcgaaag
     5641 gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg ctgattgctg
     5701 ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa gacgtgggcg
     5761 tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt aaaaacaaac
     5821 acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa ggcgaaacag
     5881 cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa gtgaattatg
     5941 gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt ggcgtgctga
     6001 gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc ctcgaaaact
     6061 atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg ggtgccgtag
     6121 cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc actatggaaa
     6181 acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc tggtatgccg
     6241 tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa gccctgaaag
     6301 acgcgcagac tccgggtagc ctggaagttc tgttccaggg gcccatgggc caggatatgg
     6361 tctcgccgcc cccgcccatt gccgatgagc cgctcaccgt gaacacgggc atctacctga
     6421 tcgagtgcta cagcctcgac gacaaagcag aaaccttcaa ggtgaatgcc tttttaagtt
     6481 tgagctggaa ggacaggcgc ctggcgtttg atccggtccg cagcggcgtt cgcgtcaaaa
     6541 cctacgaacc cgaagcgatc tggataccgg agatccgctt tgtcaatgtc gaaaatgccc
     6601 gcgacgccga tgtggtggat atttcggtaa gcccggacgg gacggtacag tatctggaac
     6661 gcttctctgc ccgagttctc tcgccgctgg attttcgccg ctatcctttt gattcgcaga
     6721 ctttacacat ttacttgatc gtccgttcgg tcgacacccg caatatcgtg cttgcggtcg
     6781 acctagaaaa agtgggcaaa aacgacgatg tgttcttgac gggatgggat atcgaaagct
     6841 ttaccgccgt ggtgaagcct gcaaacttcg ctctggaaga caggcttgaa tcgaaactgg
     6901 actaccagtt gcggatttct cgtcaatatt tttcgtacat tcccaacatc attttgccga
     6961 tgctgtttat tttatttatc tcctggactg ccttctggtc tacttcttac gaagcgaatg
     7021 taacgctggt ggtctcgacg cttatcgccc acatcgcctt caatattctg gtagaaacca
     7081 atcttccaaa gaccccctac atgacctaca cgggggcaat tatttttatg atttacctat
     7141 tttattttgt ggccgtcatt gaagttaccg ttcagcatta tctaaaagtt gagagccagc
     7201 ccgccagggc ggcgtcgatt acacgcgcct ctcgcatcgc gtttcccgtt gtattcttgc
     7261 tggcaaatat tattctggca tttcttttct ttggatttta gctcgagcac caccaccacc
     7321 accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
     7381 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     7441 tgaaaggagg aactatatcc gga
//
