LOCUS       sequence-85589-e        5137 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding multiplexed 4x gRNAs. This is a modified form of 
            the original plasmid described in the paper (Construct 19). 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 5137)
  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 5137)
  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-gRNA3-28-gRNA4-28-gRNA5-28-gRNA6-28-pA
FEATURES             Location/Qualifiers
     source          1..5137
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     226..245
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(464..568)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      595..1050
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(682..701)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     892..913
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     enhancer        1239..1542
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1543..1746
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1697..1717
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1775..1784
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1781..2458
                     /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(1961..1981)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2373..2396
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    2628..2647
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     misc_RNA        2682..2757
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    2766..2785
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     misc_RNA        2818..2893
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    2902..2921
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     misc_RNA        2954..3029
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    3038..3057
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     misc_RNA        3090..3165
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    3174..3193
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     primer_bind     complement(3398..3417)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3442..3489
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     complement(3810..3827)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3981..4569)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4061..4080)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4740..5137,1..394,395..463))
                     /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"
ORIGIN
        1 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
       61 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
      121 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
      181 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
      241 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
      301 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
      361 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
      421 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
      481 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
      541 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgcgc
      601 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
      661 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
      721 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
      781 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
      841 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
      901 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
      961 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     1021 attttaacaa aatattaacg tttacaattt cccattcgcc attcaggctg cgcaactgtt
     1081 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gcccaagcta ccatgataag
     1141 taagtaatat taaggtacgg gaggtacttg gaggtaccta gttattaata gtaatcaatt
     1201 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     1261 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     1321 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     1381 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     1441 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     1501 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
     1561 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
     1621 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
     1681 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
     1741 agagctggtt tagtgaaccg tcagatccga gctcgccacc atggcctcct ccgagaacgt
     1801 catcaccgag ttcatgcgct tcaaggtgcg catggagggc accgtgaacg gccacgagtt
     1861 cgagatcgag ggcgagggcg agggccgccc ctacgagggc cacaacaccg tgaagctgaa
     1921 ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccccc agttccagta
     1981 cggctccaag gtgtacgtga agcaccccgc cgacatcccc gactacaaga agctgtcctt
     2041 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggcgaccgt
     2101 gacccaggac tcctccctgc aggacggctg cttcatctac aaggtgaagt tcatcggcgt
     2161 gaacttcccc tccgacggcc ccgtgatgca gaagaagacc atgggctggg aggcctccac
     2221 cgagcgcctg tacccccgcg acggcgtgct gaagggcgag acccacaagg ccctgaagct
     2281 gaaggacggc ggccactacc tggtggagtt caagtccatc tacatggcca agaagcccgt
     2341 gcagctgccc ggctactact acgtggacgc caagctggac atcacctccc acaacgagga
     2401 ctacaccatc gtggagcagt acgagcgcac cgagggccgc caccacctgt tcctgtagta
     2461 aaccggtgat tcgtcagtag ggttgtaaag gtttttcttt tcctgagaaa acaacctttt
     2521 gttttctcag gttttgcttt ttggcctttc cctagcttta aaaaaaaaaa agcaaaactc
     2581 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgagtt cactgccgta
     2641 taggcagcta agaaagctag cgtgtactct ctgaggtgct cgttttagag ctagaaatag
     2701 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     2761 ttttcgttca ctgccgtata ggcagctaag aaaaggtgac gcagataaga accagttgtt
     2821 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
     2881 accgagtcgg tgcttttttt cgttcactgc cgtataggca gctaagaaac agggcatcaa
     2941 gtcagccatc agcgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
     3001 aacttgaaaa agtggcaccg agtcggtgct ttttttcgtt cactgccgta taggcagcta
     3061 agaaaagtcg gagtcaccct cctggaaacg ttttagagct agaaatagca agttaaaata
     3121 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttcgttcact
     3181 gccgtatagg cagctaagaa acccgggtaa gcgggactct ggggttcgaa atgaccgacc
     3241 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     3301 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     3361 tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac
     3421 cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt
     3481 cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga
     3541 gacgccattg gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt
     3601 cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag
     3661 ctcgagtatg tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg
     3721 cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag
     3781 gacaggtgcc ggcagcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     3841 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     3901 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     3961 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4021 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4081 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4141 gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt
     4201 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4261 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4321 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4381 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     4441 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4501 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4561 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4621 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     4681 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     4741 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     4801 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     4861 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     4921 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     4981 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5041 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5101 agctccggtt cccaacgatc aaggcgagtt acatgat
//
