LOCUS       sequence-102261-        8740 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA (no RNA aptamer addition) with COM-KRAB effector for mammalian
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8740)
  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 8740)
  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: pJZC77
FEATURES             Location/Qualifiers
     source          1..8740
                     /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        3182..3485
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3486..3689
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3726..3746
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4011..4196
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     misc_feature    4236..4809
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4403..4420)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4630..4649
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4810..5520
                     /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(4955..4973)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5223..5242
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(5540..5573)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5629..6217
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5682..5702)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6100..6111)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6220..6236)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6221..6237)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6746..6926
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6988..7576)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7068..7087)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7747..8538,8539..8607))
                     /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     8370..8389
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8608..8712)
                     /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 gttggcatac ttctgcctgc tggggagcct ggtttaagag ctatgctgga aacagcatag
     3001 caagtttaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     3061 ttttctcgag tactaggatc cattaggcgg ccgcgtggat aaccgtatta ccgccatgca
     3121 ttagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     3181 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     3241 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     3301 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     3361 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     3421 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     3481 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
     3541 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
     3601 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
     3661 tacggtggga ggtctatata agcagagctg gtttagtgaa ccgtcagatc cgctagccca
     3721 ccatgcccaa aaagaaaaga aaagtgggta gtatgaaatc aattcgctgt aaaaactgca
     3781 acaaactgtt atttaaggcg gatagttttg atcacattga aatcaggtgt ccgcgttgca
     3841 aacgtcacat cataatgctg aatgcctgcg agcatcccac ggagaaacat tgtgggaaaa
     3901 gagaaaaaat cacgcattct gacgaaaccg tgcgttatgg ctccactagt gggcacaagc
     3961 ttaatggcgg tggcggaggg atggatgcta agtcactaac tgcctggtcc cggacactgg
     4021 tgaccttcaa ggatgtattt gtggacttca ccagggagga gtggaagctg ctggacactg
     4081 ctcagcagat cgtgtacaga aatgtgatgc tggagaacta taagaacctg gtttccttgg
     4141 gttatcagct tactaagcca gatgtgatcc tccggttgga gaagggagaa gagccctagc
     4201 ccgggtcgag catgcatcta gggcggccaa ttccgcccct ctccctcccc cccccctaac
     4261 gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc
     4321 accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg
     4381 agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg
     4441 aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc
     4501 aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa
     4561 gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa
     4621 agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta
     4681 ccccattgta tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg
     4741 aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca
     4801 cgatgataaa tggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg
     4861 cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag
     4921 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc
     4981 cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac
     5041 gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag
     5101 tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc
     5161 ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac
     5221 ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc
     5281 gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac
     5341 tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc
     5401 tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa
     5461 cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagtaa
     5521 gaattcgtcg agggacctaa taacttcgta tagcatacat tatacgaagt tatacatgtt
     5581 taagggttcc ggttccacta ggtacaattc gatatcaagc ttatcgataa tcaacctctg
     5641 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     5701 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     5761 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     5821 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     5881 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     5941 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     6001 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc
     6061 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     6121 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     6181 cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgtcg acctcgatcg
     6241 agacctagaa aaacatggag caatcacaag tagcaataca gcagctacca atgctgattg
     6301 tgcctggcta gaagcacaag aggaggagga ggtgggtttt ccagtcacac ctcaggtacc
     6361 tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg
     6421 gggactggaa gggctaattc actcccaacg aagacaagat atccttgatc tgtggatcta
     6481 ccacacacaa ggctacttcc ctgattggca gaactacaca ccagggccag ggatcagata
     6541 tccactgacc tttggatggt gctacaagct agtaccagtt gagcaagaga aggtagaaga
     6601 agccaatgaa ggagagaaca cccgcttgtt acaccctgtg agcctgcatg ggatggatga
     6661 cccggagaga gaagtattag agtggaggtt tgacagccgc ctagcatttc atcacatggc
     6721 ccgagagctg catccggact gtactgggtc tctctggtta gaccagatct gagcctggga
     6781 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
     6841 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     6901 ttagtcagtg tggaaaatct ctagcagcat gtgagcaaaa ggccagcaaa aggccaggaa
     6961 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     7021 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     7081 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     7141 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     7201 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     7261 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     7321 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     7381 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     7441 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     7501 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     7561 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     7621 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     7681 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     7741 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     7801 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     7861 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     7921 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     7981 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     8041 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     8101 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     8161 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     8221 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     8281 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     8341 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     8401 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     8461 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     8521 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     8581 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     8641 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     8701 aggggttccg cgcacatttc cccgaaaagt gccacctgac
//
