LOCUS       sequence-85587-e        4781 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding the Triplex/gRNA architecture.This is a modified 
            form of the original plasmid described in the paper (Construct 3). 
            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 4781)
  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 4781)
  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-28-gRNA1-28-pA
FEATURES             Location/Qualifiers
     source          1..4781
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(108..125)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(279..867)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(359..378)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1038..1829,1830..1898))
                     /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     1661..1680
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1899..2003)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2030..2485
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2117..2136)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2327..2348
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     enhancer        2674..2977
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2978..3181
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3132..3152
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3210..3219
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3216..3893
                     /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(3396..3416)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     3808..3831
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    4063..4082
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     misc_RNA        4111..4186
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    4195..4214
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     primer_bind     complement(4477..4496)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4521..4568
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct
       61 ttatcatgca actcgtagga caggtgccgg cagcgctctt ccgcttcctc gctcactgac
      121 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
      181 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
      241 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
      301 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
      361 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
      421 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca
      481 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
      541 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
      601 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
      661 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
      721 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
      781 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
      841 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
      901 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
      961 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     1021 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     1081 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     1141 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     1201 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     1261 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     1321 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     1381 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     1441 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     1501 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     1561 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     1621 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     1681 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     1741 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     1801 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     1861 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     1921 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     1981 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgcgccctgt
     2041 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     2101 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     2161 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     2221 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     2281 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     2341 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     2401 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     2461 aacaaaatat taacgtttac aatttcccat tcgccattca ggctgcgcaa ctgttgggaa
     2521 gggcgatcgg tgcgggcctc ttcgctatta cgccagccca agctaccatg ataagtaagt
     2581 aatattaagg tacgggaggt acttggaggt acctagttat taatagtaat caattacggg
     2641 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
     2701 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
     2761 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     2821 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
     2881 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
     2941 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
     3001 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
     3061 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
     3121 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
     3181 tggtttagtg aaccgtcaga tccgagctcg ccaccatggc ctcctccgag aacgtcatca
     3241 ccgagttcat gcgcttcaag gtgcgcatgg agggcaccgt gaacggccac gagttcgaga
     3301 tcgagggcga gggcgagggc cgcccctacg agggccacaa caccgtgaag ctgaaggtga
     3361 ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc cccccagttc cagtacggct
     3421 ccaaggtgta cgtgaagcac cccgccgaca tccccgacta caagaagctg tccttccccg
     3481 agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggcg accgtgaccc
     3541 aggactcctc cctgcaggac ggctgcttca tctacaaggt gaagttcatc ggcgtgaact
     3601 tcccctccga cggccccgtg atgcagaaga agaccatggg ctgggaggcc tccaccgagc
     3661 gcctgtaccc ccgcgacggc gtgctgaagg gcgagaccca caaggccctg aagctgaagg
     3721 acggcggcca ctacctggtg gagttcaagt ccatctacat ggccaagaag cccgtgcagc
     3781 tgcccggcta ctactacgtg gacgccaagc tggacatcac ctcccacaac gaggactaca
     3841 ccatcgtgga gcagtacgag cgcaccgagg gccgccacca cctgttcctg tagtaaaccg
     3901 gtgattcgtc agtagggttg taaaggtttt tcttttcctg agaaaacaac cttttgtttt
     3961 ctcaggtttt gctttttggc ctttccctag ctttaaaaaa aaaaaagcaa aactcaccga
     4021 ggcagttcca taggatggca agatcctggt attggtctgc gagttcactg ccgtataggc
     4081 agctaagaaa tagtcgcgtg tagcgaagca gttttagagc tagaaatagc aagttaaaat
     4141 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttcgttcac
     4201 tgccgtatag gcagctaaga aacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     4261 cagcgcatca accccgggat gctagctaag cgggactctg gggttcgaaa tgaccgacca
     4321 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     4381 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     4441 gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc
     4501 ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc
     4561 gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag
     4621 acgccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc
     4681 gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagc
     4741 tcgagtatgt cgaccgatgc ccttgagagc cttcaaccca g
//
