LOCUS       sequence-102250-        8812 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA (no RNA aptamer addition) with MCP-VP64 effector for mammalian
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8812)
  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 8812)
  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: pJZC32
FEATURES             Location/Qualifiers
     source          1..8812
                     /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"
     enhancer        3176..3479
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3480..3683
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3720..3740
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4113..4262
                     /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    4308..4881
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4475..4492)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4702..4721
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4882..5592
                     /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(5027..5045)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5295..5314
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(5612..5645)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5701..6289
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5754..5774)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6172..6183)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6292..6308)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6293..6309)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6818..6998
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7060..7648)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7140..7159)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7819..8610,8611..8679))
                     /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     8442..8461
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8680..8784)
                     /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 ctttttttct
     3061 cgagtactag gatccattag gcggccgcgt ggataaccgt attaccgcca tgcattagtt
     3121 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     3181 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     3241 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     3301 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
     3361 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     3421 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
     3481 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
     3541 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     3601 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
     3661 gggaggtcta tataagcaga gctggtttag tgaaccgtca gatccgctag cccaccatgc
     3721 ccaaaaagaa aagaaaagtg ggtagtatgg cttctaactt tactcagttc gttctcgtcg
     3781 acaatggcgg aactggcgac gtgactgtcg ccccaagcaa cttcgctaac gggatcgctg
     3841 aatggatcag ctctaactcg cgttcacagg cttacaaagt aacctgtagc gttcgtcaga
     3901 gctctgcgca gaatcgcaaa tacaccatca aagtcgaggt gcctaaaggc gcctggcgtt
     3961 cgtacttaaa tatggaacta accattccaa ttttcgccac gaattccgac tgcgagctta
     4021 ttgttaaggc aatgcaaggt ctcctaaaag atggaaaccc gattccctca gcaatcgcag
     4081 caaactccgg catctacggc tccggacgag ccgatgctct cgacgatttt gacctcgata
     4141 tgctgggatc cgacgctctc gatgacttcg accttgacat gctggggagt gacgctctgg
     4201 acgacttcga tctcgacatg ctgggctccg acgccctgga tgacttcgat ctggacatgc
     4261 tcatcaactg acccgggtcg agcatgcatc tagggcggcc aattccgccc ctctccctcc
     4321 ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat
     4381 atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct
     4441 gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg
     4501 ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta
     4561 gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag
     4621 ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg
     4681 atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat
     4741 gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca
     4801 tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc
     4861 ctttgaaaaa cacgatgata aatggtgagc aagggcgagg aggataacat ggccatcatc
     4921 aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag
     4981 atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg
     5041 accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc
     5101 tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc
     5161 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc
     5221 caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac
     5281 ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag
     5341 cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag
     5401 gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag
     5461 ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac
     5521 accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag
     5581 ctgtacaagt aagaattcgt cgagggacct aataacttcg tatagcatac attatacgaa
     5641 gttatacatg tttaagggtt ccggttccac taggtacaat tcgatatcaa gcttatcgat
     5701 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     5761 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     5821 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     5881 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     5941 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     6001 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     6061 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
     6121 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     6181 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     6241 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccgt
     6301 cgacctcgat cgagacctag aaaaacatgg agcaatcaca agtagcaata cagcagctac
     6361 caatgctgat tgtgcctggc tagaagcaca agaggaggag gaggtgggtt ttccagtcac
     6421 acctcaggta cctttaagac caatgactta caaggcagct gtagatctta gccacttttt
     6481 aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag atatccttga
     6541 tctgtggatc taccacacac aaggctactt ccctgattgg cagaactaca caccagggcc
     6601 agggatcaga tatccactga cctttggatg gtgctacaag ctagtaccag ttgagcaaga
     6661 gaaggtagaa gaagccaatg aaggagagaa cacccgcttg ttacaccctg tgagcctgca
     6721 tgggatggat gacccggaga gagaagtatt agagtggagg tttgacagcc gcctagcatt
     6781 tcatcacatg gcccgagagc tgcatccgga ctgtactggg tctctctggt tagaccagat
     6841 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     6901 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     6961 cctcagaccc ttttagtcag tgtggaaaat ctctagcagc atgtgagcaa aaggccagca
     7021 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     7081 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     7141 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     7201 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     7261 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     7321 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     7381 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     7441 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     7501 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     7561 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     7621 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     7681 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     7741 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     7801 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     7861 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     7921 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     7981 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     8041 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     8101 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     8161 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     8221 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     8281 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     8341 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     8401 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     8461 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     8521 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     8581 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     8641 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     8701 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     8761 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg ac
//
