LOCUS       sequence-113829-        8933 bp    DNA     circular SYN 14-APR-2025
DEFINITION  sgRNA + 2XPP7 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 8933)
  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 8933)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: pJZC43
FEATURES             Location/Qualifiers
     source          1..8933
                     /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      3131..3150
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     enhancer        3282..3585
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3586..3789
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3826..3846
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4234..4383
                     /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    4429..5002
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4596..4613)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4823..4842
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5003..5713
                     /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(5148..5166)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5416..5435
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(5733..5766)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5822..6410
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5875..5895)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6293..6304)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6413..6429)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6414..6430)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6939..7119
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7181..7769)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7261..7280)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7940..8731,8732..8800))
                     /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     8563..8582
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8801..8905)
                     /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 cgggagctaa
     3061 ggagtttata tggaaaccct tagcctgctg cgtaaggagt ttatatggaa acccttacgc
     3121 agcagttccc tttttttgtt ttttatgtct ctgcagagtt cggtctcgag tactaggatc
     3181 cattaggcgg ccgcgtggat aaccgtatta ccgccatgca ttagttatta atagtaatca
     3241 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     3301 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     3361 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     3421 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     3481 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     3541 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
     3601 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     3661 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     3721 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     3781 agcagagctg gtttagtgaa ccgtcagatc cgctagccca ccatgcccaa aaagaaaaga
     3841 aaagtgggta gtatgtccaa aaccatcgtt ctttcggtcg gcgaggctac tcgcactctg
     3901 actgagatcc agtccaccgc agaccgtcag atcttcgaag agaaggtcgg gcctctggtg
     3961 ggtcggctgc gcctcacggc ttcgctccgt caaaacggag ccaagaccgc gtatcgcgtc
     4021 aacctaaaac tggatcaggc ggacgtcgtt gattccggac ttccgaaagt gcgctacact
     4081 caggtatggt cgcacgacgt gacaatcgtt gcgaatagca ccgaggcctc gcgcaaatcg
     4141 ttgtacgatt tgaccaagtc cctcgtcgcg acctcgcagg tcgaagatct tgtcgtcaac
     4201 cttgtgccgc tgggccgtgg ctccggacga gccgatgctc tcgacgattt tgacctcgat
     4261 atgctgggat ccgacgctct cgatgacttc gaccttgaca tgctggggag tgacgctctg
     4321 gacgacttcg atctcgacat gctgggctcc gacgccctgg atgacttcga tctggacatg
     4381 ctcatcaact gacccgggtc gagcatgcat ctagggcggc caattccgcc cctctccctc
     4441 ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta
     4501 tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc
     4561 tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct
     4621 gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt
     4681 agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa
     4741 gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg
     4801 gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg ggctgaagga
     4861 tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac
     4921 atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg acgtggtttt
     4981 cctttgaaaa acacgatgat aaatggtgag caagggcgag gaggataaca tggccatcat
     5041 caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga
     5101 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
     5161 gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg
     5221 ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc
     5281 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac
     5341 ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa
     5401 cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga
     5461 gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa
     5521 ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca
     5581 gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta
     5641 caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga
     5701 gctgtacaag taagaattcg tcgagggacc taataacttc gtatagcata cattatacga
     5761 agttatacat gtttaagggt tccggttcca ctaggtacaa ttcgatatca agcttatcga
     5821 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     5881 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     5941 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     6001 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     6061 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     6121 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     6181 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     6241 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     6301 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     6361 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg
     6421 tcgacctcga tcgagaccta gaaaaacatg gagcaatcac aagtagcaat acagcagcta
     6481 ccaatgctga ttgtgcctgg ctagaagcac aagaggagga ggaggtgggt tttccagtca
     6541 cacctcaggt acctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt
     6601 taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa gatatccttg
     6661 atctgtggat ctaccacaca caaggctact tccctgattg gcagaactac acaccagggc
     6721 cagggatcag atatccactg acctttggat ggtgctacaa gctagtacca gttgagcaag
     6781 agaaggtaga agaagccaat gaaggagaga acacccgctt gttacaccct gtgagcctgc
     6841 atgggatgga tgacccggag agagaagtat tagagtggag gtttgacagc cgcctagcat
     6901 ttcatcacat ggcccgagag ctgcatccgg actgtactgg gtctctctgg ttagaccaga
     6961 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
     7021 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
     7081 ccctcagacc cttttagtca gtgtggaaaa tctctagcag catgtgagca aaaggccagc
     7141 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     7201 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     7261 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     7321 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     7381 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     7441 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     7501 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     7561 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     7621 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     7681 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     7741 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     7801 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     7861 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     7921 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     7981 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     8041 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     8101 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     8161 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     8221 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     8281 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     8341 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     8401 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     8461 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     8521 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     8581 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     8641 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     8701 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     8761 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     8821 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     8881 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gac
//
