LOCUS       sequence-102251-        8864 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA + 1x MS2 with MCP-VP64 effector for mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8864)
  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 8864)
  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: pJZC25
FEATURES             Location/Qualifiers
     source          1..8864
                     /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"
     misc_RNA        3056..3074
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     terminator      3077..3096
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     enhancer        3228..3531
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3532..3735
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3772..3792
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4165..4314
                     /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    4360..4933
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4527..4544)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4754..4773
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4934..5644
                     /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(5079..5097)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5347..5366
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(5664..5697)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5753..6341
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5806..5826)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6224..6235)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6344..6360)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6345..6361)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6870..7050
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7112..7700)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7192..7211)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7871..8662,8663..8731))
                     /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     8494..8513
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8732..8836)
                     /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 cgcgcacatg
     3061 aggatcaccc atgtgctttt tttgtttttt atgtctctgc agagttcggt ctcgagtact
     3121 aggatccatt aggcggccgc gtggataacc gtattaccgc catgcattag ttattaatag
     3181 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
     3241 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
     3301 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
     3361 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
     3421 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
     3481 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
     3541 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
     3601 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
     3661 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
     3721 tatataagca gagctggttt agtgaaccgt cagatccgct agcccaccat gcccaaaaag
     3781 aaaagaaaag tgggtagtat ggcttctaac tttactcagt tcgttctcgt cgacaatggc
     3841 ggaactggcg acgtgactgt cgccccaagc aacttcgcta acgggatcgc tgaatggatc
     3901 agctctaact cgcgttcaca ggcttacaaa gtaacctgta gcgttcgtca gagctctgcg
     3961 cagaatcgca aatacaccat caaagtcgag gtgcctaaag gcgcctggcg ttcgtactta
     4021 aatatggaac taaccattcc aattttcgcc acgaattccg actgcgagct tattgttaag
     4081 gcaatgcaag gtctcctaaa agatggaaac ccgattccct cagcaatcgc agcaaactcc
     4141 ggcatctacg gctccggacg agccgatgct ctcgacgatt ttgacctcga tatgctggga
     4201 tccgacgctc tcgatgactt cgaccttgac atgctgggga gtgacgctct ggacgacttc
     4261 gatctcgaca tgctgggctc cgacgccctg gatgacttcg atctggacat gctcatcaac
     4321 tgacccgggt cgagcatgca tctagggcgg ccaattccgc ccctctccct cccccccccc
     4381 taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt
     4441 ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt
     4501 gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt
     4561 cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct
     4621 ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt
     4681 ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt
     4741 ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa
     4801 ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta
     4861 gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa
     4921 aacacgatga taaatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt
     4981 catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg
     5041 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
     5101 tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc
     5161 ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt
     5221 caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc
     5281 ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc
     5341 cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta
     5401 ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg
     5461 ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg
     5521 cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt
     5581 ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa
     5641 gtaagaattc gtcgagggac ctaataactt cgtatagcat acattatacg aagttataca
     5701 tgtttaaggg ttccggttcc actaggtaca attcgatatc aagcttatcg ataatcaacc
     5761 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     5821 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     5881 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     5941 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     6001 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     6061 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     6121 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt
     6181 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     6241 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     6301 ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc gtcgacctcg
     6361 atcgagacct agaaaaacat ggagcaatca caagtagcaa tacagcagct accaatgctg
     6421 attgtgcctg gctagaagca caagaggagg aggaggtggg ttttccagtc acacctcagg
     6481 tacctttaag accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa
     6541 aggggggact ggaagggcta attcactccc aacgaagaca agatatcctt gatctgtgga
     6601 tctaccacac acaaggctac ttccctgatt ggcagaacta cacaccaggg ccagggatca
     6661 gatatccact gacctttgga tggtgctaca agctagtacc agttgagcaa gagaaggtag
     6721 aagaagccaa tgaaggagag aacacccgct tgttacaccc tgtgagcctg catgggatgg
     6781 atgacccgga gagagaagta ttagagtgga ggtttgacag ccgcctagca tttcatcaca
     6841 tggcccgaga gctgcatccg gactgtactg ggtctctctg gttagaccag atctgagcct
     6901 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
     6961 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
     7021 ccttttagtc agtgtggaaa atctctagca gcatgtgagc aaaaggccag caaaaggcca
     7081 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     7141 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     7201 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     7261 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     7321 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     7381 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     7441 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     7501 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     7561 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     7621 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     7681 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     7741 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     7801 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     7861 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     7921 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     7981 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     8041 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     8101 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     8161 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     8221 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     8281 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     8341 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     8401 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     8461 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     8521 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     8581 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     8641 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     8701 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     8761 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     8821 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgac
//
