LOCUS       sequence-409420-        7548 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TFB2M protein expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7548)
  TITLE     Patel lab plasmids
REFERENCE   2  (bases 1 to 7548)
  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"
COMMENT     Sequence Label: mt deltaN42 TFB2M
FEATURES             Location/Qualifiers
     source          1..7548
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(71..88)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(98..115)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             134..156
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    171..195
                     /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)."
     primer_bind     complement(195..214)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(196..214)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(298..317)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        523..600
                     /gene="lacI"
                     /label=lacI promoter
     CDS             601..1683
                     /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     complement(620..639)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2492..2683
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2677..2699)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2785..2924
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2840..2859
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2939..2956)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3110..3698)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3190..3209)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3809..3828)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3820..4635
                     /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             3820..4635
                     /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     complement(3893..3912)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(4727..5182)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4864..4885)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5076..5095
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(5219..5266)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     5262..5280
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(5333..5350)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(6443..6463)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(join(6515..7548,1..67))
                     /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
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     complement(6518..6541)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
ORIGIN
        1 cagcgagacc gttatagcct ttatcgccgt taatccagat taccagttta ccttcttcga
       61 ttttcatgtg gctgccgcgc ggcaccaggc cgctgctgtg atgatgatga tgatggctgc
      121 tgcccatggt atatctcctt cttaaagtta aacaaaatta tttctagagg ggaattgtta
      181 tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag atctcgatcc
      241 tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct
      301 atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt
      361 gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct
      421 tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct
      481 tcctaatgca ggagtcgcat aagggagagc gtcgagatcc cggacaccat cgaatggcgc
      541 aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat
      601 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt
      661 tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg
      721 gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag
      781 tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc
      841 gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa
      901 cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt
      961 gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc
     1021 actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt
     1081 ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag
     1141 caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc
     1201 tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg
     1261 agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact
     1321 gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc
     1381 gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca
     1441 tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc
     1501 gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc
     1561 gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc
     1621 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     1681 tgagcgcaac gcaattaatg taagttagct cactcattag gcaccgggat ctcgaccgat
     1741 gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt
     1801 cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct
     1861 ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct
     1921 tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa
     1981 acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccccacggg tgcgcatgat
     2041 cgtgctcctg tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa
     2101 tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg
     2161 agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc
     2221 agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg
     2281 aacacctaca tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc
     2341 ccgccgcatc cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc
     2401 atcatcagta acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat
     2461 gaacagaaat cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa
     2521 catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga
     2581 cgcggatgaa caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg
     2641 cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     2701 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     2761 agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt
     2821 gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccattgcggt
     2881 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct
     2941 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3001 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     3061 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3121 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3181 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3241 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3301 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3361 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3421 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3481 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3541 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcgtaaaaa
     3601 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3661 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3721 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgaacaata
     3781 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
     3841 acgtcttgct ctaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
     3901 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc
     3961 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
     4021 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
     4081 atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa aacagcattc
     4141 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
     4201 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt
     4261 cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
     4321 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca
     4381 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
     4441 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
     4501 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
     4561 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
     4621 gatgagtttt tctaagaatt aattcatgag cggatacata tttgaatgta tttagaaaaa
     4681 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt
     4741 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     4801 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     4861 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     4921 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     4981 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     5041 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     5101 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     5161 aatgcgccgc tacagggcgc gtcccattcg ccaatccgga tatagttcct cctttcagca
     5221 aaaaacccct caagacccgt ttagaggccc caaggggtta tgctagttat tgctcagcgg
     5281 tggcagcagc caactcagct tcctttcggg ctttgttagc agccggatct cagtggtggt
     5341 ggtggtggtg ctcgagtgcg gccgcaagct tctacctatc ttccagggtt tcatcataca
     5401 gccatttata agcacaatct ttggaacgct ctatagtttc aaaaagtgtt ttgaagtctt
     5461 gagggtgcat gttaactact ttctcatcct cctgttttcc tatttgcatc aatatatctc
     5521 tcgcatcaag tggagtcaat gaacgtaagt ggtctattac agtggcgctg cgcctcccaa
     5581 aacagtgctt taacaagtga aaaaatatat tatagttcat aggtgttaag ttcttggtaa
     5641 ataaattttg acgaggaatc atttgaataa gatacagctt ttgttgtaat tggtctaata
     5701 attccctacg ctttgggttt tccagcggcc ctttccgggt gtatatatca aatgatgacc
     5761 aaggctccat gtgcagaacc ttaatctcac aagctaattg ccagataaca cttaatacat
     5821 gatacaagtc tggatttcca ggatctgcca ttagtttctg gaattctttt tcaccaataa
     5881 acatatttac ttctattcgt ccaaatttat atatagaagt acaggaatac aagtcatatg
     5941 cgagtttcca aagtgccctt ttctcacctc tacttgggaa cattccaact acttttaaag
     6001 ggatgtctgc tgtccaagga actgcttcta ttcccaaatt cttaaagagc cctcgagaag
     6061 acatagcagg tggttttatt actccaccac ttctaggatc tagtttaaag aagtcacagt
     6121 gaatcactcg tagttttcca tccagatttt ttcctaagga ctccaaatgt ggaataaaag
     6181 ttttgtcact ttcgagcgca accactttgg caccagcttc aagtaatgcc tgagtcagga
     6241 ttccaggacc tggattgcac tccagcagta ggtgtggagg tctacttggt tttcccaaat
     6301 agatttgcgc cagggtctca gccaatctcc gatcggttac gtaacgctta aagtctaagc
     6361 tggccttaga cgccttcctt ggcggattcc tgaaatccgg ttcgggccac agctgcggag
     6421 aggagtcaga gagcccgcta gcaccttgga aatataagtt ttcatccccg aggttgttgt
     6481 tattgttatt gttgttgttg ttcgagctcg aattagtctg cgcgtctttc agggcttcat
     6541 cgacagtctg acgaccgctg gcggcgttga tcaccgcagt acgcacggca taccagaaag
     6601 cggacatctg cgggatgttc ggcatgattt cacctttctg ggcgttttcc atagtggcgg
     6661 caatccgcgg atctttcacc aactcttcct cgtaagactt cagcgctacg gcacccagcg
     6721 gtttgtcttt attaaccgct tccagacctt catcagtcag cagatagttt tcgaggaact
     6781 cttttgccag ctctttgttc ggactggcgg cgttaatacc tgcgctcagc acaccaacga
     6841 acggtttgga tggttgaccc ttgaaggtcg gcagtaccgt tacaccataa ttcactttgc
     6901 tggtgtcgat gttggaccat gcccacgggc cgttgatggt catcgctgtt tcgcctttat
     6961 taaaggcagc ttctgcgatg gagtaatcgg tgtctgcatt catgtgtttg tttttaatca
     7021 ggtcaaccag gaaggtcaga cccgctttcg cgccagcgtt atccacgccc acgtctttaa
     7081 tgtcgtactt gccgttttca tacttgaacg cataaccccc gtcagcagca atcagcggcc
     7141 aggtgaagta cggttcttgc aggttgaaca tcagcgcgct cttacctttc gctttcagtt
     7201 ctttatccag cgccgggatc tcttcccagg tttttggcgg gttcggcagc agatctttgt
     7261 tataaatcag cgataacgct tcaacagcga tcgggtaagc aatcagcttg ccgttgtaac
     7321 gtacggcatc ccaggtaaac ggatacagct tgtcctggaa cgctttgtcc ggggtgattt
     7381 cagccaacag gccagattga gcgtagccac caaagcggtc gtgtgcccag aagataatgt
     7441 cagggccatc gccagttgcc gcaacctgtg ggaatttctc ttccagttta tccggatgct
     7501 caacggtgac tttaattccg gtatctttct cgaatttctt accgactt
//
