LOCUS       sequence-102267-        8338 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA + 2x MS2(wt+f6) with MCP-VP64 effector for mammalian cells, 
            marked by BFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8338)
  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 8338)
  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: pJZC116
FEATURES             Location/Qualifiers
     source          1..8338
                     /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        3060..3078
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     terminator      3110..3129
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     enhancer        3261..3564
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3565..3768
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3805..3825
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4198..4347
                     /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"
     CDS             4363..4419
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4420..5118
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(5138..5171)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5227..5815
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5280..5300)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5698..5709)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5818..5834)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5819..5835)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6344..6524
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6586..7174)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6666..6685)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7345..8136,8137..8205))
                     /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     7968..7987
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8206..8310)
                     /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 gttggcagac gcgaggaagg agggcgcgtt taagagctat gctggaaaca gcatagcaag
     3001 tttaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcgggagca
     3061 catgaggatc acccatgtgc gactcccaca gtcactgggg agtcttccct ttttttgttt
     3121 tttatgtctc tgcagagttc ggtctcgagt actaggatcc attaggcggc cgcgtggata
     3181 accgtattac cgccatgcat tagttattaa tagtaatcaa ttacggggtc attagttcat
     3241 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
     3301 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     3361 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     3421 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     3481 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     3541 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
     3601 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     3661 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
     3721 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac
     3781 cgtcagatcc gctagcccac catgcccaaa aagaaaagaa aagtgggtag tatggcttct
     3841 aactttactc agttcgttct cgtcgacaat ggcggaactg gcgacgtgac tgtcgcccca
     3901 agcaacttcg ctaacgggat cgctgaatgg atcagctcta actcgcgttc acaggcttac
     3961 aaagtaacct gtagcgttcg tcagagctct gcgcagaatc gcaaatacac catcaaagtc
     4021 gaggtgccta aaggcgcctg gcgttcgtac ttaaatatgg aactaaccat tccaattttc
     4081 gccacgaatt ccgactgcga gcttattgtt aaggcaatgc aaggtctcct aaaagatgga
     4141 aacccgattc cctcagcaat cgcagcaaac tccggcatct acggctccgg acgagccgat
     4201 gctctcgacg attttgacct cgatatgctg ggatccgacg ctctcgatga cttcgacctt
     4261 gacatgctgg ggagtgacgc tctggacgac ttcgatctcg acatgctggg ctccgacgcc
     4321 ctggatgact tcgatctgga catgctcatc aacggcagcg gcgctactaa cttcagcctg
     4381 ctgaagcagg ctggagacgt ggaggagaac cctggaccta gcgagctgat taaggagaac
     4441 atgcacatga agctgtacat ggagggcacc gtggacaacc atcacttcaa gtgcacatcc
     4501 gagggcgaag gcaagcccta cgagggcacc cagaccatga gaatcaaggt ggtcgagggc
     4561 ggccctctcc ccttcgcctt cgacatcctg gctactagct tcctctacgg cagcaagacc
     4621 ttcatcaacc acacccaggg catccccgac ttcttcaagc agtccttccc tgagggcttc
     4681 acatgggaga gagtcaccac atacgaagac gggggcgtgc tgaccgctac ccaggacacc
     4741 agcctccagg acggctgcct catctacaac gtcaagatca gaggggtgaa cttcacatcc
     4801 aacggccctg tgatgcagaa gaaaacactc ggctgggagg ccttcaccga gacgctgtac
     4861 cccgctgacg gcggcctgga aggcagaaac gacatggccc tgaagctcgt gggcgggagc
     4921 catctgatcg caaacatcaa gaccacatat agatccaaga aacccgctaa gaacctcaag
     4981 atgcctggcg tctactatgt ggactacaga ctggaaagaa tcaaggaggc caacaacgag
     5041 acctacgtcg agcagcacga ggtggcagtg gccagatact gcgacctccc tagcaaactg
     5101 gggcacaagc ttaattaaga attcgtcgag ggacctaata acttcgtata gcatacatta
     5161 tacgaagtta tacatgttta agggttccgg ttccactagg tacaattcga tatcaagctt
     5221 atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     5281 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     5341 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     5401 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     5461 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     5521 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     5581 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     5641 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     5701 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     5761 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga
     5821 taccgtcgac ctcgatcgag acctagaaaa acatggagca atcacaagta gcaatacagc
     5881 agctaccaat gctgattgtg cctggctaga agcacaagag gaggaggagg tgggttttcc
     5941 agtcacacct caggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca
     6001 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatat
     6061 ccttgatctg tggatctacc acacacaagg ctacttccct gattggcaga actacacacc
     6121 agggccaggg atcagatatc cactgacctt tggatggtgc tacaagctag taccagttga
     6181 gcaagagaag gtagaagaag ccaatgaagg agagaacacc cgcttgttac accctgtgag
     6241 cctgcatggg atggatgacc cggagagaga agtattagag tggaggtttg acagccgcct
     6301 agcatttcat cacatggccc gagagctgca tccggactgt actgggtctc tctggttaga
     6361 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     6421 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     6481 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagcatgt gagcaaaagg
     6541 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     6601 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     6661 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     6721 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     6781 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     6841 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     6901 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     6961 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     7021 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     7081 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     7141 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     7201 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     7261 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     7321 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     7381 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     7441 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     7501 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     7561 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     7621 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     7681 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     7741 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     7801 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     7861 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     7921 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     7981 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     8041 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     8101 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     8161 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     8221 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     8281 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgac
//
