LOCUS       Exported                7652 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  sgRNA for mammalian cells with mCherry marker.
ACCESSION   .
VERSION     .
KEYWORDS    pJZC103
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7652)
  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 7652)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7652
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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    2825..2858
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     2862..2882
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     enhancer        3175..3478
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3479..3682
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3725..4432
                     /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(3867..3885)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4135..4154
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    4452..4485
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    4541..5129
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4594..4614)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5012..5023)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5132..5148)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5133..5149)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5658..5838
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(5900..6488)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5980..5999)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6659..7519)
                     /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     7282..7301
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7520..7624)
                     /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 gttggaatag ctcagaggcc gagggtttaa gagctatgct ggaaacagca tagcaagttt
     3001 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttctc
     3061 gagtactagg atccattagg cggccgcgtg gataaccgta ttaccgccat gcattagtta
     3121 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
     3181 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
     3241 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
     3301 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     3361 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
     3421 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     3481 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
     3541 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
     3601 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
     3661 ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctagc ccaccatgcc
     3721 cggggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa
     3781 ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg
     3841 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc
     3901 cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca
     3961 ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg
     4021 cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga
     4081 cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt
     4141 aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg
     4201 cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc
     4261 tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt
     4321 caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga
     4381 acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt aagaattcgt
     4441 cgagggacct aataacttcg tatagcatac attatacgaa gttatacatg tttaagggtt
     4501 ccggttccac taggtacaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa
     4561 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     4621 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     4681 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     4741 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     4801 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     4861 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     4921 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat
     4981 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     5041 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
     5101 tcggatctcc ctttgggccg cctccccgca tcgataccgt cgacctcgat cgagacctag
     5161 aaaaacatgg agcaatcaca agtagcaata cagcagctac caatgctgat tgtgcctggc
     5221 tagaagcaca agaggaggag gaggtgggtt ttccagtcac acctcaggta cctttaagac
     5281 caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg
     5341 aagggctaat tcactcccaa cgaagacaag atatccttga tctgtggatc taccacacac
     5401 aaggctactt ccctgattgg cagaactaca caccagggcc agggatcaga tatccactga
     5461 cctttggatg gtgctacaag ctagtaccag ttgagcaaga gaaggtagaa gaagccaatg
     5521 aaggagagaa cacccgcttg ttacaccctg tgagcctgca tgggatggat gacccggaga
     5581 gagaagtatt agagtggagg tttgacagcc gcctagcatt tcatcacatg gcccgagagc
     5641 tgcatccgga ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg
     5701 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
     5761 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
     5821 tgtggaaaat ctctagcagc atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5881 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5941 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     6001 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     6061 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     6121 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     6181 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6241 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6301 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     6361 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6421 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6481 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6541 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6601 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6661 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6721 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6781 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6841 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6901 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6961 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     7021 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     7081 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     7141 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     7201 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7261 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     7321 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7381 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7441 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7501 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7561 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7621 cgcgcacatt tccccgaaaa gtgccacctg ac
//
