LOCUS       sequence-197614-        6140 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for both MS2-P65-HSF1 activator helper complex 
            and sgRNA with two MS2 loops at tetraloop and stemloop 2 contains 
            BsaI sites for insertion of spacer sequences..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6140)
  AUTHORS   Kulcsar PI, Talas A, Huszar K, Ligeti Z, Toth E, Weinhardt N, Fodor 
            E, Welker E
  TITLE     Crossing enhanced and high fidelity SpCas9 nucleases to optimize 
            specificity and cleavage.
  JOURNAL   Genome Biol. 2017 Oct 6;18(1):190. doi: 10.1186/s13059-017-1318-8.
  PUBMED    28985763
REFERENCE   2  (bases 1 to 6140)
  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: "Genome 
            Biol."; date: "2017-10-6"; volume: "18(1):190. doi"; pages: " 
            10.1186/s13059-017-1318-8"
COMMENT     Sequence Label: U6-sgRNA(MS2)_EF1a-MS2-P65-HSF1
FEATURES             Location/Qualifiers
     source          1..6140
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        287..305
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        357..375
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     promoter        507..1685
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          738..1676
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1633..1653
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1692..1701
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1701..2087
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /label=MS2-N55K
                     /note="binds with high affinity to a specific stem-loop 
                     structure in the viral RNA (Lim et al., 1994)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     CDS             2142..2162
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2178..2720
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             2745..3116
                     /codon_start=1
                     /product="C-terminal activation domain from the human heat 
                     shock transcription factor HSF1"
                     /label=HSF1
                     /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR
                     PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED
                     PTISLLTGSEPPKAKDPTVS"
     primer_bind     complement(3181..3198)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3187..3394
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3403..3543
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3403..3532
                     /label=AAV2 ITR
     rep_origin      3618..4073
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3705..3724)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3915..3936
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4090..4109)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4209..4231
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4269..4287)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4355..4459
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4460..4528,4529..5320)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4678..4697)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5491..6079
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5980..5999
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg gagaccgata tcggtctccg ttttagagct aggccaacat gaggatcacc
      301 catgtctgca gggcctagca agttaaaata aggctagtcc gttatcaact tggccaacat
      361 gaggatcacc catgtctgca gggccaagtg gcaccgagtc ggtgcttttt ttgaattgtt
      421 aattaatgca aagatggata aagttttaaa cagagaggaa tctttgcagc taatggacct
      481 tctaggtctt gaaaggagtg ggaattggct ccggtgcccg tcagtgggca gagcgcacat
      541 cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt gcctagagaa
      601 ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg
      661 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt
      721 ttgccgccag aacacaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg
      781 gttatggccc ttgcgtgcct tgaattactt ccacctggct gcagtacgtg attcttgatc
      841 ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt
      901 cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt
      961 ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa aatttttgat
     1021 gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc caagatctgc
     1081 acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca
     1141 catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc
     1201 aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg
     1261 cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg cttcccggcc
     1321 ctgctgcagg gagctcaaaa tgaaggacgc ggcgctcggg agagcgggcg ggtgagtcac
     1381 ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac tccacggagt
     1441 accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg tcgtctttag
     1501 gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg gagactgaag
     1561 ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt gagtttggat
     1621 cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt
     1681 cgtgacgtac ggccaccatg gcttcaaact ttactcagtt cgtgctcgtg gacaatggtg
     1741 ggacagggga tgtgacagtg gctccttcta atttcgctaa tggggtggca gagtggatca
     1801 gctccaactc acggagccag gcctacaagg tgacatgcag cgtcaggcag tctagtgccc
     1861 agaagagaaa gtataccatc aaggtggagg tccccaaagt ggctacccag acagtgggcg
     1921 gagtcgaact gcctgtcgcc gcttggaggt cctacctgaa catggagctc actatcccaa
     1981 ttttcgctac caattctgac tgtgaactca tcgtgaaggc aatgcagggg ctcctcaaag
     2041 acggtaatcc tatcccttcc gccatcgccg ctaactcagg tatctacagc gctggaggag
     2101 gtggaagcgg aggaggagga agcggaggag gaggtagcgg acctaagaaa aagaggaagg
     2161 tggcggccgc tggatcccct tcagggcaga tcagcaacca ggccctggct ctggccccta
     2221 gctccgctcc agtgctggcc cagactatgg tgccctctag tgctatggtg cctctggccc
     2281 agccacctgc tccagcccct gtgctgaccc caggaccacc ccagtcactg agcgctccag
     2341 tgcccaagtc tacacaggcc ggcgagggga ctctgagtga agctctgctg cacctgcagt
     2401 tcgacgctga tgaggacctg ggagctctgc tggggaacag caccgatccc ggagtgttca
     2461 cagatctggc ctccgtggac aactctgagt ttcagcagct gctgaatcag ggcgtgtcca
     2521 tgtctcatag tacagccgaa ccaatgctga tggagtaccc cgaagccatt acccggctgg
     2581 tgaccggcag ccagcggccc cccgaccccg ctccaactcc cctgggaacc agcggcctgc
     2641 ctaatgggct gtccggagat gaagacttct caagcatcgc tgatatggac tttagtgccc
     2701 tgctgtcaca gatttcctct agtgggcagg gaggaggtgg aagcggcttc agcgtggaca
     2761 ccagtgccct gctggacctg ttcagcccct cggtgaccgt gcccgacatg agcctgcctg
     2821 accttgacag cagcctggcc agtatccaag agctcctgtc tccccaggag ccccccaggc
     2881 ctcccgaggc agagaacagc agcccggatt cagggaagca gctggtgcac tacacagcgc
     2941 agccgctgtt cctgctggac cccggctccg tggacaccgg gagcaacgac ctgccggtgc
     3001 tgtttgagct gggagagggc tcctacttct ccgaagggga cggcttcgcc gaggacccca
     3061 ccatctccct gctgacaggc tcggagcctc ccaaagccaa ggaccccact gtctccgcta
     3121 gttagggggc ctaggtgtac aagtaagaat tctagacaat tcctagagct cgctgatcag
     3181 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     3241 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     3301 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     3361 aggattggga agagaatagc aggcatgctg gggagcggcc gcaggaaccc ctagtgatgg
     3421 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     3481 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     3541 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     3601 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3661 tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt
     3721 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3781 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     3841 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     3901 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg
     3961 ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct
     4021 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     4081 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     4141 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     4201 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     4261 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     4321 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     4381 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     4441 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     4501 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     4561 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     4621 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     4681 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     4741 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     4801 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     4861 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     4921 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     4981 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     5041 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     5101 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     5161 agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     5221 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     5281 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     5341 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     5401 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     5461 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     5521 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     5581 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     5641 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     5701 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     5761 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     5821 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     5881 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     5941 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     6001 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     6061 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     6121 ctggcctttt gctcacatgt
//
