LOCUS       sequence-85588-e        4732 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding the Ribozyme/gRNA architecture. This is a modified 
            form of the original plasmid described in the paper (Construct 13). 
            mKate2 was replaced with dsRed2 because of distribution issues..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4732)
  AUTHORS   Nissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu TK
  TITLE     Multiplexed and Programmable Regulation of Gene Networks with an 
            Integrated RNA and CRISPR/Cas Toolkit in Human Cells.
  JOURNAL   Mol Cell. 2014 May 14. pii: S1097-2765(14)00355-4. doi: 
            10.1016/j.molcel.2014.04.022.
  PUBMED    24837679
REFERENCE   2  (bases 1 to 4732)
  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: "Mol Cell. 
            2014 May 14. pii: S1097-2765(14)00355-4. doi: 
            10.1016/j.molcel.2014.04.022."
COMMENT     Sequence Label: CMVp-dsRed2-Triplex-HHRibo-gRNA1-HDVRibo-pA
FEATURES             Location/Qualifiers
     source          1..4732
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(589..1380,1381..1449))
                     /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     1212..1231
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1450..1554)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1581..2036
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1668..1687)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1878..1899
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     enhancer        2225..2528
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2529..2732
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2683..2703
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2761..2770
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2767..3444
                     /codon_start=1
                     /product="improved tetrameric variant of DsRed fluorescent 
                     protein"
                     /label=DsRed2
                     /note="mammalian codon-optimized"
                     /translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     primer_bind     complement(2947..2967)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     3359..3382
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     ncRNA           3570..3606
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        3627..3702
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           3707..3774
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(3979..3998)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4023..4070
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     complement(4391..4408)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(4562..4732,1..418))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4642..4661)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt
       61 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
      121 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
      181 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
      241 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
      301 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
      361 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
      421 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
      481 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
      541 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
      601 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
      661 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
      721 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
      781 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
      841 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
      901 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
      961 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     1021 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     1081 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     1141 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     1201 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     1261 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     1321 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     1381 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     1441 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     1501 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     1561 tccccgaaaa gtgccacctg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     1621 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     1681 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     1741 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     1801 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     1861 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     1921 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     1981 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caatttccca
     2041 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
     2101 acgccagccc aagctaccat gataagtaag taatattaag gtacgggagg tacttggagg
     2161 tacctagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
     2221 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     2281 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     2341 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
     2401 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
     2461 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
     2521 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
     2581 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
     2641 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
     2701 gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgagctc
     2761 gccaccatgg cctcctccga gaacgtcatc accgagttca tgcgcttcaa ggtgcgcatg
     2821 gagggcaccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac
     2881 gagggccaca acaccgtgaa gctgaaggtg accaagggcg gccccctgcc cttcgcctgg
     2941 gacatcctgt ccccccagtt ccagtacggc tccaaggtgt acgtgaagca ccccgccgac
     3001 atccccgact acaagaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac
     3061 ttcgaggacg gcggcgtggc gaccgtgacc caggactcct ccctgcagga cggctgcttc
     3121 atctacaagg tgaagttcat cggcgtgaac ttcccctccg acggccccgt gatgcagaag
     3181 aagaccatgg gctgggaggc ctccaccgag cgcctgtacc cccgcgacgg cgtgctgaag
     3241 ggcgagaccc acaaggccct gaagctgaag gacggcggcc actacctggt ggagttcaag
     3301 tccatctaca tggccaagaa gcccgtgcag ctgcccggct actactacgt ggacgccaag
     3361 ctggacatca cctcccacaa cgaggactac accatcgtgg agcagtacga gcgcaccgag
     3421 ggccgccacc acctgttcct gtagtaaacc ggtgattcgt cagtagggtt gtaaaggttt
     3481 ttcttttcct gagaaaacaa ccttttgttt tctcaggttt tgctttttgg cctttcccta
     3541 gctttaaaaa aaaaaaagca aaacgactac tgatgagtcc gtgaggacga aacgagtaag
     3601 ctcgtctagt cgcgtgtagc gaagcagttt tagagctaga aatagcaagt taaaataagg
     3661 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttggcc ggcatggtcc
     3721 cagcctcctc gctggcgccg gctgggcaac atgcttcggc atggcgaatg ggaccccggg
     3781 atgctagcta agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     3841 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     3901 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     3961 ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat
     4021 gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg
     4081 ggttcggtcc cagggctggc actctgtcga taccccaccg agacgccatt ggggccaata
     4141 cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct
     4201 cgcagccaac gtcggggcgg caggccctgc catagcctca gctcgagtat gtcgaccgat
     4261 gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt
     4321 cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct
     4381 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4441 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4501 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4561 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4621 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4681 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc cc
//
