LOCUS       sequence-194419-        7792 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  recombinant expression of target protein as an MBP-His tag fusionat 
            the N-terminus of target protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7792)
  AUTHORS   Hirschi M, Johnson ZL, Lee SY
  TITLE     Visualizing multistep elevator-like transitions of a nucleoside 
            transporter.
  JOURNAL   Nature. 2017 May 4;545(7652):66-70. doi: 10.1038/nature22057. Epub 
            2017 Apr 17.
  PUBMED    28424521
REFERENCE   2  (bases 1 to 7792)
  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: 
            "10.1038/nature22057"; journalName: "Nature"; date: "2017-05-4- 4"; 
            volume: "545"; issue: "7652"; pages: "66-70"
COMMENT     Sequence Label: pET26-CNTN149S/F366A
FEATURES             Location/Qualifiers
     source          1..7792
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     sig_peptide     89..154
                     /codon_start=1
                     /gene="pelB (fragment)"
                     /product="leader peptide for secretion"
                     /label=pelB signal sequence
                     /translation="MKYLLPTAAAGLLLLAAQPAMA"
     CDS             194..223
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             227..1327
                     /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     1301..1324
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1337..1360
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2657..2674
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2727..2745)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2741..2788
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2825..3280
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2912..2931)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3122..3143
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3372..4187)
                     /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(3372..4187)
                     /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     4095..4114
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4179..4198
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4309..4897
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4798..4817
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5051..5068
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5083..5222
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5148..5167)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5308..5330
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5324..5515)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6324..7406)
                     /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     7368..7387
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7407..7484)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7690..7709
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gaaatacctg ctgccgaccg ctgctgctgg
      121 tctgctgctc ctcgctgccc agccggcgat ggcaatggat atcggaatta attcggatcc
      181 gaattcgagc tcacatcacc atcaccatca ccatcaccat caccccatga aaatcgaaga
      241 aggtaaactg gtaatctgga ttaacggcga taaaggctat aacggtctcg ctgaagtcgg
      301 taagaaattc gagaaagata ccggaattaa agtcaccgtt gagcatccgg ataaactgga
      361 agagaaattc ccacaggttg cggcaactgg cgatggccct gacattatct tctgggcaca
      421 cgaccgcttt ggtggctacg ctcaatctgg cctgttggct gaaatcaccc cggacaaagc
      481 gttccaggac aagctgtatc cgtttacctg ggatgccgta cgttacaacg gcaagctgat
      541 tgcttacccg atcgctgttg aagcgttatc gctgatttat aacaaagatc tgctgccgaa
      601 cccgccaaaa acctgggaag agatcccggc gctggataaa gaactgaaag cgaaaggtaa
      661 gagcgcgctg atgttcaacc tgcaagaacc gtacttcacc tggccgctga ttgctgctga
      721 cgggggttat gcgttcaagt atgaaaacgg caagtacgac attaaagacg tgggcgtgga
      781 taacgctggc gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa acaaacacat
      841 gaatgcagac accgattact ccatcgcaga agctgccttt aataaaggcg aaacagcgat
      901 gaccatcaac ggcccgtggg catggtccaa catcgacacc agcaaagtga attatggtgt
      961 aacggtactg ccgaccttca agggtcaacc atccaaaccg ttcgttggcg tgctgagcgc
     1021 aggtattaac gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg aaaactatct
     1081 gctgactgat gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg ccgtagcgct
     1141 gaagtcttac gaggaagagt tggcgaaaga tccacgtatt gccgccacta tggaaaacgc
     1201 ccagaaaggt gaaatcatgc cgaacatccc gcagatgtcc gctttctggt atgccgtgcg
     1261 tactgcggtg atcaacgccg ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc
     1321 gcagactccg ggtagcctgg aagttctgtt ccaggggccc gcggtaccta ggatggttgt
     1381 gctgcattcc ctgctgggta tggctgtgct gattgctatt gctgtcctgc tgtccaccga
     1441 ccgtaaagcg attaacatcc gtaccgttgc gggtgccttt ctgattcagg tcgcgctggg
     1501 cgcactggtc ctgtatgtgc cgcaaggccg cgatatgctg ggtgaagcaa gtaaaacgat
     1561 ttccaatgtg atcgcttacg gcaacaatgg tgttgacttt ctgttcggcg gtctggtctc
     1621 agaaaaaatg tttgaagtgt tcggcggtgg cggttttgtt ttcgcgctgc gtgtcctgcc
     1681 gatgattgtg tttttcagct ctctgatggc cgttctgtat tacatcggtg tcatgcagct
     1741 gctgattaaa gtgatcggcg gttttctgca aaaaatgctg ggcaccagca aagccgaaag
     1801 tatgagcgcg gcggcgagca ttttcgttgg ccagaccgaa gcaccgctgg ttgtgcgccc
     1861 gtatatccgt cgcatgacgg aaagcgaact gtttgcagtg atgtctggcg gtctggcatc
     1921 agtggctggc tcggttctgg cgggttatgt ccagatgggc gtgccgctgc cgtacctgat
     1981 tgctgcgagt tttatggcag caccgggcgg tctgctgttc gcaaaactgc tggttccgga
     2041 aaccgaacgt acgcagaatg atgccgaagt gctggcagaa aacgaagatg aaaaaccgac
     2101 caatgttatc gacgctgcgg ccagtggtgc tgtcacgggt gcgcaaattg caatcgctgt
     2161 cggtgcatcc ctgctggcat tcgtggcact gattgcaatg atcaacggca ttatcggcgg
     2221 tgtgggcggt tggtttggcc atggtgatct gaccctgcag gccattctgg gttggctgtt
     2281 ctcaccgctg gcatgggtta tcggcgtccc gtggagtgaa gctggcattg cgggttccct
     2341 gatcggccag aaagtcgtga ttaacgaatt tgtggcgtat tcggaattcg ttaaatacct
     2401 gaaaccggaa gcagctgttc aactgtcaga taccacgaaa gccattatct cgtttgcact
     2461 gtgcggtgcc gctaatctgg gcagcattgc cgtgctggtt ggcggtctgt ctatcatggc
     2521 gccgaaacgt cgcaaagacg tggcccgcct gggtattaaa gccgttgtcg caggctcact
     2581 gtcgaacctg atgagcgctg ttatcgcggg tctgtttacc ggcctgagcg gtgcgtctgt
     2641 gctgggctaa ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg
     2701 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     2761 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
     2821 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2881 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2941 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     3001 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     3061 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     3121 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     3181 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     3241 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     3301 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     3361 tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     3421 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     3481 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     3541 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     3601 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
     3661 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     3721 attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
     3781 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     3841 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     3901 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     3961 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     4021 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     4081 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     4141 tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta
     4201 ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt
     4261 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4321 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4381 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4441 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4501 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4561 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4621 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4681 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4741 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4801 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4861 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4921 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4981 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     5041 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     5101 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
     5161 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     5221 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     5281 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     5341 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     5401 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     5461 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     5521 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     5581 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     5641 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     5701 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     5761 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     5821 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     5881 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     5941 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga
     6001 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     6061 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     6121 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     6181 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     6241 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     6301 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     6361 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     6421 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     6481 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     6541 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     6601 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     6661 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     6721 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     6781 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     6841 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     6901 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     6961 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     7021 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     7081 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     7141 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     7201 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     7261 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     7321 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     7381 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     7441 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     7501 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     7561 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     7621 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     7681 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     7741 gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa at
//
