LOCUS       sequence-248771-        6677 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of MBP-CfaN fusion protein (with C-terminal 
            His5).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6677)
  TITLE     Muir Lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6677)
  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            "Unpublished"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-MBP-CfaN-His6
FEATURES             Location/Qualifiers
     source          1..6677
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     213..232
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        213..231
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    232..256
                     /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             271..293
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             304..330
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             331..1428
                     /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      1285..1294
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1750..1767
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1820..1838)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1834..1881
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1918..2373
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2005..2024)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2215..2236
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2465..3280)
                     /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(2465..3280)
                     /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     3188..3207
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3272..3291
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3402..3990
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3891..3910
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4144..4161
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4176..4315
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4241..4260)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4401..4423
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4417..4608)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5417..6499)
                     /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     6461..6480
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6500..6577)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atcgatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
      241 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacat
      301 atgtaccctt acgacgttcc tgactacgct aaaatcgaag aaggtaaact ggtaatctgg
      361 attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat
      421 accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt
      481 gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac
      541 gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat
      601 ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt
      661 gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa
      721 gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac
      781 ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag
      841 tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg
      901 ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac
      961 tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg
     1021 gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc
     1081 aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt
     1141 ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg
     1201 gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag
     1261 ttggcgaaag atccacgtat tgccgccacc atggaaaacg cccagaaagg tgaaatcatg
     1321 ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc
     1381 gccagcggtc gtcagactgt cgatgaagcc ccgaaagacg cgcagactaa tgcggaatat
     1441 tgcctgtctt acgacacaga gattctgacc gttgaatatg gattccttcc tatcggtaag
     1501 atcgtggagg aacggattga atgcacagtc tatacggtag ataaaaatgg ctttgtgtat
     1561 acacaaccta ttgctcagtg gcataaccgg ggagaacagg aagttttcga atactgctta
     1621 gaagacggtt cgattatccg tgcaacgaaa gatcacaaat ttatgacgac cgacggtcag
     1681 atgttaccga ttgatgagat tttcgaacgg gggttagacc tgaaacaagt tgatggtttg
     1741 ccgggaggtc atcaccacca tcatcactga gatccggctg ctaacaaaga ccgaaaggaa
     1801 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     1861 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
     1921 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     1981 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     2041 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     2101 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     2161 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     2221 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     2281 ggattttgcc gattttggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     2341 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     2401 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta
     2461 attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat
     2521 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt
     2581 tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac
     2641 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga
     2701 cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag
     2761 gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg
     2821 attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa
     2881 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag
     2941 gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg
     3001 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc
     3061 agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca
     3121 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc
     3181 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc
     3241 gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt
     3301 ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc
     3361 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     3421 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     3481 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     3541 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     3601 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     3661 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     3721 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     3781 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     3841 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     3901 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     3961 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4021 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     4081 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     4141 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     4201 catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg
     4261 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     4321 gcacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     4381 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     4441 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     4501 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     4561 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     4621 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     4681 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     4741 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     4801 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     4861 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     4921 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     4981 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     5041 cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc
     5101 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     5161 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     5221 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     5281 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     5341 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     5401 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5461 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     5521 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     5581 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     5641 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt
     5701 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     5761 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     5821 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     5881 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     5941 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     6001 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     6061 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     6121 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     6181 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     6241 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     6301 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     6361 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     6421 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     6481 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     6541 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     6601 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     6661 gcaaggaatg gtgcatg
//
