LOCUS       sequence-416233-        3425 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid express MS2 loop containing Cj Cas9 gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3425)
  AUTHORS   Mahata B, Cabrera A, Brenner DA, Guerra-Resendez RS, Li J, Goell J, 
            Wang K, Guo Y, Escobar M, Parthasarathy AK, Szadowski H, Bedford G, 
            Reed DR, Kim S, Hilton IB
  TITLE     Compact engineered human mechanosensitive transactivation modules 
            enable potent and versatile synthetic transcriptional control.
  JOURNAL   Nat Methods. 2023 Oct 9. doi: 10.1038/s41592-023-02036-1.
  PUBMED    37813990
REFERENCE   2  (bases 1 to 3425)
  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: "Nat 
            Methods. 2023 Oct 9. doi: 10.1038/s41592-023-02036-1."
COMMENT     Sequence Label: U6-CjCas9-MS2gRNA-Backbone
FEATURES             Location/Qualifiers
     source          1..3425
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(529..1320,1321..1389))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLHKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1152..1171
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1390..1494)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1573..2027)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1709..1730)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1921..1940
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2153..2175
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2167..2184
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2168..2184
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2191..2210
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2191..2209
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2294..2542
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2294..2314
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2465..2484
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2582..2600
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     CDS             2707..2733
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2713..2739
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     promoter        complement(2757..2775)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2758..2775)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2793..2809)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2793..2809)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2806..2828)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2817..2833
                     /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)."
     promoter        complement(join(2841..2847,2848..2865,2866..2871))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2886..2907
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3024..3041)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(3195..3425,1..358))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3275..3294)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
       61 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
      121 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
      181 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
      241 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
      301 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
      361 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
      421 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
      481 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
      541 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
      601 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
      661 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
      721 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
      781 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgtgcaacg ttgttgccat
      841 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
      901 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
      961 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     1021 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     1081 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     1141 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     1201 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     1261 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     1321 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     1381 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     1441 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     1501 tccccgaaaa gtgccacctg atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     1561 accgcatcag ggaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     1621 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     1681 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     1741 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     1801 accatcactc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     1861 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     1921 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     1981 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcca ttcgccattc
     2041 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
     2101 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
     2161 cgacgttgta aaacgacggc cagtgaattg taatacgact cactataggg cgaattgatt
     2221 acaaggatga cgacgatgaa caaaaactct ctcagaagag gatctggaat tcgatctcga
     2281 gtctagattc accgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     2341 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
     2401 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     2461 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     2521 cttgtggaaa ggacgaaaca ccgggagacg ggatcccgtc tccgttttag tccctgagcc
     2581 aacatgagga tcacccatgt ctgcagggcc gggactaaaa taaagagttt gcgggactct
     2641 gcggggttac aatcccctaa aaccgctttt tttgacattg attattgact agtcgacatc
     2701 gaattctatc cttatgacgt gcctgactat gccagcctgg gaggacgctt gagtattcta
     2761 tagtgtcacc taaatagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     2821 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     2881 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     2941 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     3001 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     3061 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     3121 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     3181 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     3241 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     3301 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     3361 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     3421 taggt
//
