LOCUS       sequence-392972-        8305 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human FMRP protein in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8305)
  AUTHORS   Edwards M, Xu M, Joseph S
  TITLE     A simple procedure for bacterial expression and purification of the 
            fragile X protein family.
  JOURNAL   Sci Rep. 2020 Sep 28;10(1):15858. doi: 10.1038/s41598-020-72984-7.
  PUBMED    32985615
REFERENCE   2  (bases 1 to 8305)
  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: "Sci Rep."; 
            date: "2020-09-28"; volume: "10(1):15858. doi"; pages: " 
            10.1038/s41598-020-72984-7"
COMMENT     Sequence Label: His6_MBP_FMRP_P451S
FEATURES             Location/Qualifiers
     source          1..8305
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      35..623
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     524..543
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     777..794
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    809..948
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(874..893)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1034..1056
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1050..1241)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2050..3132)
                     /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
                     YIPPLTTIKHDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     3094..3113
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3133..3210)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3416..3435
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3523..3542
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3523..3541
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3542..3566
                     /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             3581..3603
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3614..3631
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3656..4753
                     /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
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      4610..4619
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     4727..4750
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             4775..4795
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             6764..6781
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6834..6852)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6848..6895
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(7019..7038)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        7205..7308
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7309..7377,7378..8169)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7527..7546)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
       61 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
      121 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
      181 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
      241 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
      301 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
      361 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
      421 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
      481 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
      541 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
      601 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
      661 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
      721 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
      781 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
      841 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
      901 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
      961 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     1021 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     1081 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     1141 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     1201 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     1261 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     1321 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     1381 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     1441 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     1501 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     1561 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     1621 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     1681 gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggactgcttc
     1741 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     1801 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     1861 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     1921 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     1981 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     2041 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     2101 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     2161 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     2221 caagcggtcc acgctggttt gccccagcag gcgaaaatca tgtttgatgg tggttaacgg
     2281 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc
     2341 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     2401 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     2461 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     2521 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     2581 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     2641 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     2701 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     2761 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     2821 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     2881 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     2941 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     3001 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     3061 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     3121 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     3181 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     3241 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     3301 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     3361 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     3421 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     3481 gtccggcgta gaggatcgag atcgatctcg atcccgcgaa attaatacga ctcactatag
     3541 gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg
     3601 agatatacat atgcaccatc atcatcatca ttcttctggt gtagatctgg gtaccaaaat
     3661 cgaagaaggt aaactggtaa tctggattaa cggcgataaa ggctataacg gtctcgctga
     3721 agtcggtaag aaattcgaga aagataccgg aattaaagtc accgttgagc atccggataa
     3781 actggaagag aaattcccac aggttgcggc aactggcgat ggccctgaca ttatcttctg
     3841 ggcacacgac cgctttggtg gctacgctca atctggcctg ttggccgaaa tcaccccgga
     3901 caaagcgttc caggacaagc tgtatccgtt tacctgggat gccgtacgtt acaacggcaa
     3961 gctgattgct tacccgatcg ctgttgaagc gttatcgctg atttataaca aagatctgct
     4021 gccgaacccg ccaaaaacct gggaagagat cccggcgctg gataaagaac tgaaagcgaa
     4081 aggtaagagc gcgctgatgt tcaacctgca agaaccgtac ttcacctggc cgctgattgc
     4141 tgctgacggg ggttatgcgt tcaagtatga aaacggcaag tacgacatta aagacgtggg
     4201 cgtggataac gctggcgcga aagcgggtct gaccttcctg gttgacctga ttaaaaacaa
     4261 acacatgaat gcagacaccg attactccat cgcagaagct gcctttaata aaggcgaaac
     4321 agcgatgacc atcaacggcc cgtgggcatg gtccaacatc gacaccagca aagtgaatta
     4381 tggtgtaacg gtactgccga ccttcaaggg tcaaccatcc aaaccgttcg ttggcgtgct
     4441 gagcgcaggt attaacgccg ccagtccgaa caaagagctg gcaaaagagt tcctcgaaaa
     4501 ctatctgctg actgatgaag gtctggaagc ggttaataaa gacaaaccgc tgggtgccgt
     4561 agcgctgaag tcttacgagg aagagttggc gaaagatcca cgtattgccg ccaccatgga
     4621 aaacgcccag aaaggtgaaa tcatgccgaa catcccgcag atgtccgctt tctggtatgc
     4681 cgtgcgtact gcggtgatca acgccgccag cggtcgtcag actgtcgatg aagccctgaa
     4741 agacgcgcag actgattacg atatcccagg taccgagaac ctgtacttcc aatccaatgc
     4801 catggaagaa ctggtggttg aagtgcgtgg ttcgaatggc gcgttttaca aagccttcgt
     4861 gaaggacgtt catgaagata gcattaccgt tgcgtttgaa aacaattggc agccggatcg
     4921 ccaaatcccg tttcacgacg ttcgtttccc gccgccggtc ggttacaaca aggacatcaa
     4981 cgaatctgat gaagtggaag tttacagtcg cgcgaacgaa aaagaaccgt gctgttggtg
     5041 gctggccaaa gtccgtatga ttaagggcga attttatgtg atcgaatacg cggcctgcga
     5101 tgcgacctat aatgaaattg tcacgatcga acgcctgcgt agcgtgaacc cgaataaacc
     5161 ggccaccaag gatacgttcc ataaaattaa gctggacgtg ccggaagatc tgcgtcagat
     5221 gtgtgcaaaa gaagcagctc acaaggattt taaaaaggcg gtcggtgcct tctctgtgac
     5281 ctatgacccg gaaaactacc agctggtgat tctgtccatc aatgaagtta cctcaaaacg
     5341 cgcgcacatg ctgattgata tgcactttcg ctcgctgcgt accaaactga gcctgatcat
     5401 gcgtaacgaa gaagcatcca agcagctgga aagttcccgc caactggctt cacgttttca
     5461 tgaacagttc attgtgcgcg aagatctgat gggcctggca attggtaccc acggcgcgaa
     5521 tatccagcaa gcccgtaaag tcccgggtgt gacggctatt gatctggacg aagatacctg
     5581 cacgttccat atctacggcg aagaccaaga tgcagttaaa aaggctcgct cctttctgga
     5641 attcgccgaa gatgttattc aggtcccgcg taacctggtg ggtaaagtta tcggtaaaaa
     5701 tggcaagctg attcaagaaa tcgttgataa aagcggcgtc gtgcgcgtcc gtattgaagc
     5761 agaaaacgaa aagaacgtgc cgcaggaaga agaaatcatg ccgccgaact cactgccgtc
     5821 gaacaatagc cgcgttggtc cgaatgctcc ggaagaaaag aaacatctgg atatcaagga
     5881 aaacagcacc cacttttctc agccgaatag tacgaaggtg caacgtgtcc tggtggcatc
     5941 atcggttgtc gctggcgaat ctcagaaacc ggaactgaag gcctggcagg gtatggttcc
     6001 gtttgttttc gtcggcacca aagatagtat cgcaaacgct acggttctgc tggactatca
     6061 tctgaattac ctgaaagaag tcgatcagct gcgcctggaa cgtctgcaaa ttgacgaaca
     6121 gctgcgccaa atcggtgcaa gctctcgtcc gtctccgaac cgtaccgata aagaaaagtc
     6181 ttatgtgacg gatgacggtc agggtatggg tcgtggcagt cgtccgtatc gcaatcgtgg
     6241 tcatggtcgt cgcggtccgg gttacacctc cggtacgaac tctgaagcaa gtaacgcttc
     6301 cgaaaccgaa tcagaccacc gcgatgaact gtcagattgg tcgctggcgc cgacggaaga
     6361 agaacgtgaa tcttttctgc gtcgcggtga cggtcgtcgc cgtggcggtg gcggtcgtgg
     6421 tcagggcggt cgcggccgtg gcggtggctt caaaggcaat gatgaccata gtcgtaccga
     6481 taatcgtccg cgtaatccgc gcgaagccaa aggtcgtacc acggatggct cgctgcagat
     6541 tcgcgtggac tgtaacaatg aacgtagcgt tcacaccaaa acgctgcaaa acaccagttc
     6601 cgaaggtagc cgcctgcgta cgggcaaaga tcgtaatcag aaaaaggaaa aaccggactc
     6661 agtggatggt caacaaccgc tggtcaatgg tgtgccgtaa cattggaagt ggataacgga
     6721 tccgaattcg agctccgtcg acaagcttgc ggccgcactc gagcaccacc accaccacca
     6781 ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
     6841 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     6901 aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     6961 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     7021 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     7081 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     7141 cttgattagg gtgatggttc acattaacgc ttacaattta ggtggcactt ttcggggaaa
     7201 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     7261 gagacaataa ccctgataaa tgcttcaata atttgaaaaa ggaagagtat gagtattcaa
     7321 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     7381 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     7441 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     7501 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     7561 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     7621 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     7681 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     7741 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     7801 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg
     7861 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     7921 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     7981 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     8041 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     8101 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     8161 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     8221 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     8281 taacgtgagt tttcgttcca ctgag
//
