LOCUS       Exported                7711 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  sgRNA + 1x PP7 with mCherry for mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    pJZC39
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7711)
  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 7711)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7711
                     /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"
     terminator      3083..3102
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     enhancer        3234..3537
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3538..3741
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3784..4491
                     /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(3926..3944)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4194..4213
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    4511..4544
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    4600..5188
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4653..4673)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5071..5082)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5191..5207)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5192..5208)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5717..5897
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(5959..6547)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6039..6058)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6718..7578)
                     /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     7341..7360
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7579..7683)
                     /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 caacataagg
     3061 agtttatatg gaaaccctta tgtttttttg ttttttatgt ctctgcagag ttcggtctcg
     3121 agtactagga tccattaggc ggccgcgtgg ataaccgtat taccgccatg cattagttat
     3181 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
     3241 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
     3301 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
     3361 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
     3421 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
     3481 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
     3541 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
     3601 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
     3661 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
     3721 gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagcc caccatgccc
     3781 ggggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
     3841 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
     3901 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     3961 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
     4021 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
     4081 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac
     4141 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
     4201 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
     4261 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
     4321 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
     4381 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
     4441 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta agaattcgtc
     4501 gagggaccta ataacttcgt atagcataca ttatacgaag ttatacatgt ttaagggttc
     4561 cggttccact aggtacaatt cgatatcaag cttatcgata atcaacctct ggattacaaa
     4621 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     4681 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc
     4741 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt
     4801 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc
     4861 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc
     4921 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg
     4981 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt
     5041 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc
     5101 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt
     5161 cggatctccc tttgggccgc ctccccgcat cgataccgtc gacctcgatc gagacctaga
     5221 aaaacatgga gcaatcacaa gtagcaatac agcagctacc aatgctgatt gtgcctggct
     5281 agaagcacaa gaggaggagg aggtgggttt tccagtcaca cctcaggtac ctttaagacc
     5341 aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga
     5401 agggctaatt cactcccaac gaagacaaga tatccttgat ctgtggatct accacacaca
     5461 aggctacttc cctgattggc agaactacac accagggcca gggatcagat atccactgac
     5521 ctttggatgg tgctacaagc tagtaccagt tgagcaagag aaggtagaag aagccaatga
     5581 aggagagaac acccgcttgt tacaccctgt gagcctgcat gggatggatg acccggagag
     5641 agaagtatta gagtggaggt ttgacagccg cctagcattt catcacatgg cccgagagct
     5701 gcatccggac tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
     5761 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
     5821 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
     5881 gtggaaaatc tctagcagca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5941 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     6001 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     6061 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     6121 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     6181 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     6241 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     6301 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     6361 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     6421 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     6481 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     6541 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     6601 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     6661 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     6721 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     6781 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6841 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     6901 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6961 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     7021 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     7081 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     7141 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     7201 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     7261 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     7321 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     7381 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     7441 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     7501 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     7561 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     7621 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     7681 gcgcacattt ccccgaaaag tgccacctga c
//
