LOCUS       sequence-113832-        8885 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA + 1XPP7 with PCP-VP64 effector for mammalian cells, marked by 
            mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8885)
  AUTHORS   Zalatan JG, Lee ME, Almeida R, Gilbert LA, Whitehead EH, La Russa M,
            Tsai JC, Weissman JS, Dueber JE, Qi LS, Lim WA
  TITLE     Engineering Complex Synthetic Transcriptional Programs with CRISPR 
            RNA Scaffolds.
  JOURNAL   Cell. 2014 Dec 18. pii: S0092-8674(14)01570-0. doi: 
            10.1016/j.cell.2014.11.052.
  PUBMED    25533786
REFERENCE   2  (bases 1 to 8885)
  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: "Cell. 2014 
            Dec 18. pii: S0092-8674(14)01570-0. doi: 
            10.1016/j.cell.2014.11.052."
COMMENT     Sequence Label: pJZC42
FEATURES             Location/Qualifiers
     source          1..8885
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(56..75)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        247..626
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        628..826
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             844..1024
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1071..1196
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1695..1928
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2113..2157
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2455..2572
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(2825..2858)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2862..2882
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     terminator      3083..3102
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     enhancer        3234..3537
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3538..3741
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3778..3798
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4186..4335
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     misc_feature    4381..4954
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4548..4565)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4775..4794
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4955..5665
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(5100..5118)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5368..5387
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(5685..5718)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5774..6362
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5827..5847)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6245..6256)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6365..6381)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6366..6382)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6891..7071
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7133..7721)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7213..7232)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7892..8683,8684..8752))
                     /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     8515..8534
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8753..8857)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gcttaagcgg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
       61 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
      121 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
      181 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
      241 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      301 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      361 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      421 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      481 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      541 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      601 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      661 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      721 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      781 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt
      841 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
      901 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
      961 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     1021 agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac
     1081 gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta
     1141 cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta
     1201 ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga
     1261 aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta
     1321 atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat
     1381 cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca accctctatt
     1441 gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag
     1501 agcaaaacaa aagtaagacc accgcacagc aagcggccgg ccgcgctgat cttcagacct
     1561 ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa
     1621 attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
     1681 agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg
     1741 ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag
     1801 cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc
     1861 tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa
     1921 cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg
     1981 aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag
     2041 tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa
     2101 aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg
     2161 aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga
     2221 ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag
     2281 ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc
     2341 gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac
     2401 ggatcggcac tgcgtgcgcc aattctgcag acaaatggca gtattcatcc acaattttaa
     2461 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac
     2521 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta
     2581 ttacagggac agcagagatc cagtttggtt agtaccgggc ccgctctaga gatccgacgc
     2641 gccatctcta ggcccgcgcc ggccccctcg cacagacttg tgggagaagc tcggctactc
     2701 ccctgccccg gttaatttgc atataatatt tcctagtaac tatagaggct taatgtgcga
     2761 taaaagacag ataatctgtt ctttttaata ctagctacat tttacatgat aggcttggat
     2821 ttctataact tcgtatagca tacattatac gaagttataa acagcacaaa aggaaactca
     2881 ccctaactgt aaagtaattg tgtgttttga gactataagt atcccttgga gaaccacctt
     2941 gttggtacgt tctctatcac tgatagttta agagctatgc tggaaacagc atagcaagtt
     3001 taaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg caacataagg
     3061 agtttatatg gaaaccctta tgtttttttg ttttttatgt ctctgcagag ttcggtctcg
     3121 agtactagga tccattaggc ggccgcgtgg ataaccgtat taccgccatg cattagttat
     3181 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
     3241 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
     3301 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
     3361 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
     3421 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
     3481 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
     3541 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
     3601 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
     3661 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
     3721 gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagcc caccatgccc
     3781 aaaaagaaaa gaaaagtggg tagtatgtcc aaaaccatcg ttctttcggt cggcgaggct
     3841 actcgcactc tgactgagat ccagtccacc gcagaccgtc agatcttcga agagaaggtc
     3901 gggcctctgg tgggtcggct gcgcctcacg gcttcgctcc gtcaaaacgg agccaagacc
     3961 gcgtatcgcg tcaacctaaa actggatcag gcggacgtcg ttgattccgg acttccgaaa
     4021 gtgcgctaca ctcaggtatg gtcgcacgac gtgacaatcg ttgcgaatag caccgaggcc
     4081 tcgcgcaaat cgttgtacga tttgaccaag tccctcgtcg cgacctcgca ggtcgaagat
     4141 cttgtcgtca accttgtgcc gctgggccgt ggctccggac gagccgatgc tctcgacgat
     4201 tttgacctcg atatgctggg atccgacgct ctcgatgact tcgaccttga catgctgggg
     4261 agtgacgctc tggacgactt cgatctcgac atgctgggct ccgacgccct ggatgacttc
     4321 gatctggaca tgctcatcaa ctgacccggg tcgagcatgc atctagggcg gccaattccg
     4381 cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg
     4441 tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg
     4501 gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg
     4561 aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca
     4621 aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct
     4681 ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca
     4741 cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa
     4801 ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg
     4861 cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg
     4921 ggacgtggtt ttcctttgaa aaacacgatg ataaatggtg agcaagggcg aggaggataa
     4981 catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg
     5041 ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc
     5101 caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca
     5161 gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa
     5221 gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt
     5281 ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct
     5341 gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga
     5401 ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag
     5461 gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa
     5521 gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca
     5581 caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg
     5641 cggcatggac gagctgtaca agtaagaatt cgtcgaggga cctaataact tcgtatagca
     5701 tacattatac gaagttatac atgtttaagg gttccggttc cactaggtac aattcgatat
     5761 caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
     5821 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     5881 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
     5941 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
     6001 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
     6061 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     6121 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt
     6181 tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
     6241 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
     6301 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
     6361 gcatcgatac cgtcgacctc gatcgagacc tagaaaaaca tggagcaatc acaagtagca
     6421 atacagcagc taccaatgct gattgtgcct ggctagaagc acaagaggag gaggaggtgg
     6481 gttttccagt cacacctcag gtacctttaa gaccaatgac ttacaaggca gctgtagatc
     6541 ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac
     6601 aagatatcct tgatctgtgg atctaccaca cacaaggcta cttccctgat tggcagaact
     6661 acacaccagg gccagggatc agatatccac tgacctttgg atggtgctac aagctagtac
     6721 cagttgagca agagaaggta gaagaagcca atgaaggaga gaacacccgc ttgttacacc
     6781 ctgtgagcct gcatgggatg gatgacccgg agagagaagt attagagtgg aggtttgaca
     6841 gccgcctagc atttcatcac atggcccgag agctgcatcc ggactgtact gggtctctct
     6901 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     6961 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     7021 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agcatgtgag
     7081 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     7141 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     7201 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     7261 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     7321 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     7381 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     7441 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     7501 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     7561 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     7621 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     7681 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     7741 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     7801 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     7861 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     7921 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     7981 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     8041 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     8101 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     8161 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     8221 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     8281 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     8341 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     8401 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     8461 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     8521 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     8581 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     8641 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     8701 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     8761 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     8821 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     8881 ctgac
//
