LOCUS       sequence-427165-        5609 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His/MBP-tagged MS2-CP V75E/A81G variant in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5609)
  AUTHORS   Hemphill WO, Voong CK, Fenske R, Goodrich JA, Cech TR
  TITLE     Multiple RNA- and DNA-binding proteins exhibit direct transfer of 
            polynucleotides with implications for target-site search.
  JOURNAL   Proc Natl Acad Sci U S A. 2023 Jun 27;120(26):e2220537120. doi: 
            10.1073/pnas.2220537120. Epub 2023 Jun 20.
  PUBMED    37339225
REFERENCE   2  (bases 1 to 5609)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2023-06-27"; volume: "120(26):e2220537120. 
            doi"; pages: " 10.1073/pnas.2220537120. Epub 2023 Jun 20"
COMMENT     Sequence Label: pMALcPP-MS2cp
FEATURES             Location/Qualifiers
     source          1..5609
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..78
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             79..1161
                     /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(98..117)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1404..1409,1410..1425,1426..1432)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1440..1456
                     /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     1445..1467
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1535..1552
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     2618..2641
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2693..2716
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2726..3115
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /label=MS2
                     /note="binds to a specific stem-loop structure in the viral
                     RNA (Peabody, 1993)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQNRKYTIKVEVPKVATQTVGGEELPVAGWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     terminator      3138..3224
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3316..3343
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3371..3461
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3462..3530,3531..4322)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3680..3699)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4410..4998
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4899..4918
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5152..5169
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5352..5374
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5368..5559)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gacaccatcg aatggtgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt
       61 caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt
      121 gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg
      181 cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa
      241 caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac
      301 gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg
      361 gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt
      421 ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt
      481 gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca
      541 cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg
      601 gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg
      661 cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg
      721 gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat
      781 gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg
      841 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tttcggtagt gggatacgac
      901 gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc
      961 ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag
     1021 ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaataca
     1081 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     1141 cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc
     1201 acaattctca tgtttgacag cttatcatcg actgcacggt gcaccaatgc ttctggcgtc
     1261 aggcagccat cggaagctgt ggtatggctg tgcaggtcgt aaatcactgc ataattcgtg
     1321 tcgctcaagg cgcactcccg ttctggataa tgttttttgc gccgacatca taacggttct
     1381 ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcg gctcgtataa tgtgtggaat
     1441 tgtgagcgga taacaatttc acacaggaaa cagccagtcc gtttaggtgt tttcacgagc
     1501 aattgaccaa caaggaccat agattatgaa aatccaccat caccaccacc acgaagaagg
     1561 taaactggta atctggatta acggcgataa aggctataac ggtctcgctg aagtcggtaa
     1621 gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata aactggaaga
     1681 gaaattccca caggttgcgg caactggcga tggccctgac attatcttct gggcacacga
     1741 ccgctttggt ggctacgctc aatctggcct gttggctgaa atcaccccgg acaaagcgtt
     1801 ccaggacaag ctgtatccgt ttacctggga tgccgtacgt tacaacggca agctgattgc
     1861 ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc tgccgaaccc
     1921 gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga aaggtaagag
     1981 cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg ctgctgacgg
     2041 gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa
     2101 cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg attaaaaaca aacacatgaa
     2161 tgcagacacc gattactcca tcgcagaagc agcctttaat aaaggcgaaa cagcgatgac
     2221 catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt atggtgtaac
     2281 ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg
     2341 tattaacgcc gccagtccga acaaagagct ggcaaaagag ttcctcgaaa actatctgct
     2401 gactgatgaa ggtctggaag cggttaataa agacaaaccg ctgggtgccg tagcgctgaa
     2461 gtcttacgag gaagagttgg tgaaagatcc gcgtattgcc gccactatgg aaaacgccca
     2521 gaaaggtgaa atcatgccga acatcccgca gatgtccgct ttctggtatg ccgtgcgtac
     2581 tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat gaagccctga aagacgcgca
     2641 gactaattcg agctcgaaca acaacaacaa taacaataac aacaacctcg ggctggaagt
     2701 tctgttccag gggcccggat ccatggcttc taactttact cagttcgttc tcgtcgacaa
     2761 tggcggaact ggcgacgtga ctgtcgcccc aagcaacttc gctaacgggg tcgctgaatg
     2821 gatcagctct aactcgcgtt cacaggctta caaagtaacc tgtagcgttc gtcagagctc
     2881 tgcgcagaat cgcaaataca ccatcaaagt cgaggtgcct aaagtggcaa cccagactgt
     2941 tggtggtgaa gagcttcctg tagccggatg gcgttcgtac ctaaatatgg aactaaccat
     3001 tccaattttc gccacgaatt ccgactgcga gcttattgtt aaggcaatgc aaggtctcct
     3061 aaaagatgga aacccgattc cctcagcaat cgcagcaaac tccggcatct actgactgca
     3121 ggtaattaaa taagcttcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt
     3181 ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt
     3241 tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca
     3301 ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt ctacaaactc
     3361 tttcggtccg ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     3421 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     3481 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     3541 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     3601 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt ttcccaatga
     3661 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag
     3721 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     3781 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     3841 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     3901 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     3961 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     4021 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     4081 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     4141 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     4201 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     4261 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     4321 aactgtcaga ccaagtttac tcatatatac tttagattga tttccttagg actgagcgtc
     4381 aaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     4441 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     4501 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     4561 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     4621 gctctgctaa tcctgttacc agtgggtgct gccagtggcg ataagtcgtg tcttaccggg
     4681 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     4741 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     4801 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     4861 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     4921 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     4981 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     5041 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     5101 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     5161 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     5221 atttcacacc gcatataagg tgcactgtga ctgggtcatg gctgcgcccc gacacccgcc
     5281 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     5341 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     5401 gaggcagctg cggtaaagct catcagcgtg gtcgtgcagc gattcacaga tgtctgcctg
     5461 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     5521 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaagctca
     5581 ggccaggacc caacgctgcc cgaaattcc
//
