LOCUS       sequence-102244-        7653 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA for mammalian cells with mCherry marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7653)
  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 7653)
  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: pJZC35
FEATURES             Location/Qualifiers
     source          1..7653
                     /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             3726..4433
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(3868..3886)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4136..4155
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(4453..4486)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4542..5130
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4595..4615)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5013..5024)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5133..5149)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5134..5150)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5659..5839
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(5901..6489)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5981..6000)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6660..7451,7452..7520))
                     /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     7283..7302
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7521..7625)
                     /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 ccggggtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca
     3781 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg
     3841 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc
     3901 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc
     3961 accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc
     4021 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg
     4081 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg
     4141 taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg
     4201 gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg
     4261 ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg
     4321 tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg
     4381 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag taagaattcg
     4441 tcgagggacc taataacttc gtatagcata cattatacga agttatacat gtttaagggt
     4501 tccggttcca ctaggtacaa ttcgatatca agcttatcga taatcaacct ctggattaca
     4561 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     4621 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     4681 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     4741 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     4801 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     4861 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     4921 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga
     4981 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     5041 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     5101 gtcggatctc cctttgggcc gcctccccgc atcgataccg tcgacctcga tcgagaccta
     5161 gaaaaacatg gagcaatcac aagtagcaat acagcagcta ccaatgctga ttgtgcctgg
     5221 ctagaagcac aagaggagga ggaggtgggt tttccagtca cacctcaggt acctttaaga
     5281 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg
     5341 gaagggctaa ttcactccca acgaagacaa gatatccttg atctgtggat ctaccacaca
     5401 caaggctact tccctgattg gcagaactac acaccagggc cagggatcag atatccactg
     5461 acctttggat ggtgctacaa gctagtacca gttgagcaag agaaggtaga agaagccaat
     5521 gaaggagaga acacccgctt gttacaccct gtgagcctgc atgggatgga tgacccggag
     5581 agagaagtat tagagtggag gtttgacagc cgcctagcat ttcatcacat ggcccgagag
     5641 ctgcatccgg actgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     5701 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     5761 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     5821 gtgtggaaaa tctctagcag catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5881 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5941 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     6001 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     6061 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     6121 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     6181 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     6241 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6301 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     6361 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     6421 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6481 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6541 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6601 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     6661 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     6721 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     6781 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     6841 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6901 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6961 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     7021 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     7081 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     7141 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     7201 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     7261 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     7321 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     7381 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     7441 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     7501 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     7561 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     7621 ccgcgcacat ttccccgaaa agtgccacct gac
//
