LOCUS       sequence-322478-        5990 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express the RBD of human MSI2 in bacterial cells for protein 
            purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5990)
  AUTHORS   Clingman CC, Deveau LM, Hay SA, Genga RM, Shandilya SM, Massi F, 
            Ryder SP
  TITLE     Allosteric inhibition of a stem cell RNA-binding protein by an 
            intermediary metabolite.
  JOURNAL   Elife. 2014 Jun 16:e02848. doi: 10.7554/eLife.02848.
  PUBMED    24935936
REFERENCE   2  (bases 1 to 5990)
  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: "Elife. 2014
            Jun 16:e02848. doi: 10.7554/eLife.02848."
COMMENT     Sequence Label: pET-22HT-MSI2(8-193)
FEATURES             Location/Qualifiers
     source          1..5990
                     /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)"
     CDS             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             113..133
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             722..739
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(792..810)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      806..853
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      890..1345
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(977..996)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1187..1208
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1371..1475
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1476..1544,1545..2336)
                     /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(1694..1713)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2507..3095
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2996..3015
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3249..3266
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3281..3420
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3346..3365)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3506..3528
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3522..3713)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4522..5604)
                     /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     5566..5585
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5605..5682)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5888..5907
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcatcatcat catcatcacg gcgaaaacct
      121 gtattttcag ggcggatccg gcacctcggg cagcgccaac gactcccagc acgaccccgg
      181 taaaatgttt atcggtggac tgagctggca gacctcacca gatagcctta gagactattt
      241 tagcaaattt ggagaaatta gagaatgtat ggtcatgaga gatcccacta cgaaacgctc
      301 cagaggcttc ggtttcgtca cgttcgcaga cccagcaagt gtagataaag tattaggtca
      361 gccccaccat gagttagatt ccaagacgat tgaccccaaa gttgcatttc ctcgtcgagc
      421 gcaacccaag atggtcacga gaacaaagaa aatatttgta ggcgggttat ctgcgaacac
      481 agtagtggaa gatgtaaagc aatatttcga gcagtttggc aaggtggaag atgcaatgct
      541 gatgtttgat aaaactacca acaggcacag agggtttggc tttgtcactt ttgagaatga
      601 agatgttgtg gagaaagtct gtgagattca tttccatgaa atcaataata aaatggtaga
      661 atgtaagaaa gctcagccga aagaagtcat gttcccatga aagcttgcgg ccgcactcga
      721 gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt
      781 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
      841 gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt
      901 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
      961 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     1021 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     1081 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     1141 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     1201 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     1261 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     1321 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
     1381 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     1441 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     1501 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     1561 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     1621 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     1681 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     1741 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     1801 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     1861 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     1921 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     1981 aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc
     2041 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     2101 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     2161 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     2221 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     2281 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     2341 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     2401 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     2461 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     2521 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     2581 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     2641 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     2701 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     2761 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     2821 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     2881 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     2941 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     3001 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     3061 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     3121 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     3181 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     3241 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     3301 ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga
     3361 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     3421 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     3481 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     3541 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     3601 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     3661 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     3721 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     3781 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     3841 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     3901 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     3961 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     4021 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     4081 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     4141 cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata
     4201 atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg
     4261 gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag
     4321 cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata
     4381 aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg
     4441 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     4501 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4561 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     4621 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     4681 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     4741 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     4801 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     4861 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     4921 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     4981 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     5041 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     5101 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     5161 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     5221 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     5281 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     5341 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     5401 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     5461 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     5521 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     5581 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     5641 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     5701 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     5761 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     5821 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     5881 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     5941 cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
//
