LOCUS       sequence-240663-        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"
     enhancer        145..448
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        449..652
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     603..623
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      681..690
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             687..1364
                     /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(867..887)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1279..1302
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     ncRNA           1490..1526
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        1547..1622
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           1627..1694
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(1899..1918)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1943..1990
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     complement(2311..2328)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2482..3070)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2562..2581)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3241..4032,4033..4101))
                     /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     3864..3883
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4102..4206)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4233..4688
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4320..4339)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4530..4551
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gtgcgggcct cttcgctatt acgccagccc aagctaccat gataagtaag taatattaag
       61 gtacgggagg tacttggagg tacctagtta ttaatagtaa tcaattacgg ggtcattagt
      121 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
      181 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
      241 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
      301 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
      361 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
      421 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
      481 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
      541 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
      601 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt
      661 gaaccgtcag atccgagctc gccaccatgg cctcctccga gaacgtcatc accgagttca
      721 tgcgcttcaa ggtgcgcatg gagggcaccg tgaacggcca cgagttcgag atcgagggcg
      781 agggcgaggg ccgcccctac gagggccaca acaccgtgaa gctgaaggtg accaagggcg
      841 gccccctgcc cttcgcctgg gacatcctgt ccccccagtt ccagtacggc tccaaggtgt
      901 acgtgaagca ccccgccgac atccccgact acaagaagct gtccttcccc gagggcttca
      961 agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggc gaccgtgacc caggactcct
     1021 ccctgcagga cggctgcttc atctacaagg tgaagttcat cggcgtgaac ttcccctccg
     1081 acggccccgt gatgcagaag aagaccatgg gctgggaggc ctccaccgag cgcctgtacc
     1141 cccgcgacgg cgtgctgaag ggcgagaccc acaaggccct gaagctgaag gacggcggcc
     1201 actacctggt ggagttcaag tccatctaca tggccaagaa gcccgtgcag ctgcccggct
     1261 actactacgt ggacgccaag ctggacatca cctcccacaa cgaggactac accatcgtgg
     1321 agcagtacga gcgcaccgag ggccgccacc acctgttcct gtagtaaacc ggtgattcgt
     1381 cagtagggtt gtaaaggttt ttcttttcct gagaaaacaa ccttttgttt tctcaggttt
     1441 tgctttttgg cctttcccta gctttaaaaa aaaaaaagca aaacgactac tgatgagtcc
     1501 gtgaggacga aacgagtaag ctcgtctagt cgcgtgtagc gaagcagttt tagagctaga
     1561 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
     1621 gcttttggcc ggcatggtcc cagcctcctc gctggcgccg gctgggcaac atgcttcggc
     1681 atggcgaatg ggaccccggg atgctagcta agcgggactc tggggttcga aatgaccgac
     1741 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     1801 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     1861 atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata
     1921 ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg
     1981 tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg
     2041 agacgccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt
     2101 tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca
     2161 gctcgagtat gtcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg
     2221 gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta
     2281 ggacaggtgc cggcagcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     2341 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     2401 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     2461 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     2521 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     2581 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     2641 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     2701 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     2761 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     2821 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     2881 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     2941 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     3001 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     3061 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     3121 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     3181 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     3241 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     3301 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     3361 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     3421 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     3481 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     3541 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     3601 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     3661 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     3721 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     3781 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     3841 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     3901 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     3961 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     4021 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     4081 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     4141 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     4201 tccgcgcaca tttccccgaa aagtgccacc tgacgcgccc tgtagcggcg cattaagcgc
     4261 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     4321 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     4381 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     4441 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     4501 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     4561 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     4621 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     4681 tacaatttgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cg
//
