LOCUS       sequence-248773-        7429 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of CfaC-MBP fusion protein (with N-terminal 
            His6-Sumo).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7429)
  TITLE     Muir Lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7429)
  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            "Unpublished"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-His6-Sumo-CfaC-MBP
FEATURES             Location/Qualifiers
     source          1..7429
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     171..190
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     274..293
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        274..292
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    293..317
                     /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             332..354
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             373..390
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             400..417
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             421..714
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             829..855
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             856..1953
                     /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="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEAPKDAQT"
     regulatory      1810..1819
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2203..2221)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2217..2264
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2439..2444,2445..2461,2462..2467))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2494..2512)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2580..2684
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2685..2753,2754..3545)
                     /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(2903..2922)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3716..4304
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4205..4224
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4458..4475
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4490..4629
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4555..4574)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4715..4737
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4731..4922)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6234..7316)
                     /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     7278..7297
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7317..7394)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
       61 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
      121 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
      181 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
      241 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt
      301 gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatatacc
      361 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat
      421 atgtcggact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct
      481 gagactcaca tcaatttaaa ggtgtccgat ggatcttcag agatcttctt caagatcaaa
      541 aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg
      601 gactccttaa gattcttgta cgacggtatt agaattcaag ctgatcagac ccctgaagat
      661 ttggacatgg aggataacga tattattgag gctcacagag aacagattgg tggtgtcaag
      721 atcattagtc gtaagagtct gggcactcaa aacgtctacg atattggagt agagaaggac
      781 cataatttcc tgcttaagaa tgggctggtg gcctctaact gcttcaacta cccttacgac
      841 gttcctgact acgctaaaat cgaagaaggt aaactggtaa tctggattaa cggcgataaa
      901 ggctataacg gtctcgctga agtcggtaag aaattcgaga aagataccgg aattaaagtc
      961 accgttgagc atccggataa actggaagag aaattcccac aggttgcggc aactggcgat
     1021 ggccctgaca ttatcttctg ggcacacgac cgctttggtg gctacgctca atctggcctg
     1081 ttggctgaaa tcaccccgga caaagcgttc caggacaagc tgtatccgtt tacctgggat
     1141 gccgtacgtt acaacggcaa gctgattgct tacccgatcg ctgttgaagc gttatcgctg
     1201 atttataaca aagatctgct gccgaacccg ccaaaaacct gggaagagat cccggcgctg
     1261 gataaagaac tgaaagcgaa aggtaagagc gcgctgatgt tcaacctgca agaaccgtac
     1321 ttcacctggc cgctgattgc tgctgacggg ggttatgcgt tcaagtatga aaacggcaag
     1381 tacgacatta aagacgtggg cgtggataac gctggcgcga aagcgggtct gaccttcctg
     1441 gttgacctga ttaaaaacaa acacatgaat gcagacaccg attactccat cgcagaagct
     1501 gcctttaata aaggcgaaac agcgatgacc atcaacggcc cgtgggcatg gtccaacatc
     1561 gacaccagca aagtgaatta tggtgtaacg gtactgccga ccttcaaggg tcaaccatcc
     1621 aaaccgttcg ttggcgtgct gagcgcaggt attaacgccg ccagtccgaa caaagagctg
     1681 gcaaaagagt tcctcgaaaa ctatctgctg actgatgaag gtctggaagc ggttaataaa
     1741 gacaaaccgc tgggtgccgt agcgctgaag tcttacgagg aagagttggc gaaagatcca
     1801 cgtattgccg ccaccatgga aaacgcccag aaaggtgaaa tcatgccgaa catcccgcag
     1861 atgtccgctt tctggtatgc cgtgcgtact gcggtgatca acgccgccag cggtcgtcag
     1921 actgtcgatg aagccccgaa agacgcgcag actaattaag gcaacagatt cctgtggtga
     1981 tttaatgttc ctaatgaaat ggaaagacac agatgaagct gacctggttc ttgcaaaaga
     2041 agctaatgtg aaatgtccac aaattgtgat agcattttat gaagagagac tgacatggca
     2101 tgcatatcct gaggatgcgg aaaacaaaga gaaagaaaca gcaaagagct aaggatccgg
     2161 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
     2221 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
     2281 tatccggata tcccgcaaga ggcccggcag taccggcata accaagccta tgcctacagc
     2341 atccagggtg acggtgccga ggatgacgat gagcgcattg ttagatttca tacacggtgc
     2401 ctgactgcgt tagcaattta actgtgataa actaccgcat taaagcttat cgatgataag
     2461 ctgtcaaaca tgagaattct tgaagacgaa agggcctcgt gatacgccta tttttatagg
     2521 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     2581 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     2641 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     2701 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     2761 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     2821 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     2881 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc
     2941 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     3001 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     3061 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     3121 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     3181 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa
     3241 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     3301 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     3361 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     3421 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     3481 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     3541 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     3601 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     3661 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3721 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3781 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3841 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     3901 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3961 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4021 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4081 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4141 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4201 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4261 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4321 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     4381 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     4441 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     4501 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     4561 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     4621 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     4681 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     4741 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     4801 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     4861 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     4921 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     4981 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     5041 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     5101 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     5161 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     5221 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     5281 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     5341 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ctaggaccca
     5401 acgctgcccg agatgcgccg cgtgcggctg ctggagatgg cggacgcgat ggatatgttc
     5461 tgccaagggt tggtttgcgc attcacagtt ctccgcaaga attgattggc tccaattctt
     5521 ggagtggtga atccgttagc gaggtgccgc cggcttccat tcaggtcgag gtggcccggc
     5581 tccatgcacc gcgacgcaac gcggggaggc agacaaggta tagggcggcg cctacaatcc
     5641 atgccaaccc gttccatgtg ctcgccgagg cggcataaat cgccgtgacg atcagcggtc
     5701 caatgatcga agttaggctg gtaagagccg cgagcgatcc ttgaagctgt ccctgatggt
     5761 cgtcatctac ctgcctggac agcatggcct gcaacgcggg catcccgatg ccgccggaag
     5821 cgagaagaat cataatgggg aaggccatcc agcctcgcgt cgcgaacgcc agcaagacgt
     5881 agcccagcgc gtcggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     5941 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     6001 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     6061 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     6121 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     6181 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     6241 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     6301 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     6361 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     6421 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     6481 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     6541 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     6601 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     6661 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     6721 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     6781 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     6841 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     6901 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     6961 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     7021 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     7081 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     7141 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     7201 tccacttttt cccgcgtttt agcagaaacg tggctggcct ggttcaccac gcgggaaacg
     7261 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     7321 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     7381 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgc
//
