LOCUS       sequence-102265-        8779 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA + 1x COM with COM-KRAB effector for mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8779)
  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 8779)
  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: pJZC74
FEATURES             Location/Qualifiers
     source          1..8779
                     /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"
     terminator      3070..3089
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     enhancer        3221..3524
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3525..3728
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3765..3785
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4050..4235
                     /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    4275..4848
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4442..4459)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4669..4688
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4849..5559
                     /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(4994..5012)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5262..5281
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(5579..5612)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5668..6256
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5721..5741)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6139..6150)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6259..6275)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6260..6276)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6785..6965
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7027..7615)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7107..7126)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7786..8577,8578..8646))
                     /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     8409..8428
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8647..8751)
                     /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 ctgaatgcct
     3061 gcgagcatct ttttttgttt tttatgtctc tgcagagttc ggtctcgagt actaggatcc
     3121 attaggcggc cgcgtggata accgtattac cgccatgcat tagttattaa tagtaatcaa
     3181 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     3241 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     3301 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     3361 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     3421 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     3481 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
     3541 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
     3601 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     3661 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     3721 gcagagctgg tttagtgaac cgtcagatcc gctagcccac catgcccaaa aagaaaagaa
     3781 aagtgggtag tatgaaatca attcgctgta aaaactgcaa caaactgtta tttaaggcgg
     3841 atagttttga tcacattgaa atcaggtgtc cgcgttgcaa acgtcacatc ataatgctga
     3901 atgcctgcga gcatcccacg gagaaacatt gtgggaaaag agaaaaaatc acgcattctg
     3961 acgaaaccgt gcgttatggc tccactagtg ggcacaagct taatggcggt ggcggaggga
     4021 tggatgctaa gtcactaact gcctggtccc ggacactggt gaccttcaag gatgtatttg
     4081 tggacttcac cagggaggag tggaagctgc tggacactgc tcagcagatc gtgtacagaa
     4141 atgtgatgct ggagaactat aagaacctgg tttccttggg ttatcagctt actaagccag
     4201 atgtgatcct ccggttggag aagggagaag agccctagcc cgggtcgagc atgcatctag
     4261 ggcggccaat tccgcccctc tccctccccc ccccctaacg ttactggccg aagccgcttg
     4321 gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc
     4381 aatgtgaggg cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc
     4441 cctctcgcca aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa
     4501 gcttcttgaa gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct
     4561 ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca
     4621 caaccccagt gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca
     4681 agcgtattca acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat
     4741 ctggggcctc ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc
     4801 cccccgaacc acggggacgt ggttttcctt tgaaaaacac gatgataaat ggtgagcaag
     4861 ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag
     4921 ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
     4981 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac
     5041 atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc
     5101 cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
     5161 gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc
     5221 tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag
     5281 accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc
     5341 gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc
     5401 acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg
     5461 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc
     5521 cgccactcca ccggcggcat ggacgagctg tacaagtaag aattcgtcga gggacctaat
     5581 aacttcgtat agcatacatt atacgaagtt atacatgttt aagggttccg gttccactag
     5641 gtacaattcg atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga
     5701 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     5761 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     5821 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
     5881 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
     5941 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
     6001 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
     6061 aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg
     6121 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
     6181 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
     6241 tgggccgcct ccccgcatcg ataccgtcga cctcgatcga gacctagaaa aacatggagc
     6301 aatcacaagt agcaatacag cagctaccaa tgctgattgt gcctggctag aagcacaaga
     6361 ggaggaggag gtgggttttc cagtcacacc tcaggtacct ttaagaccaa tgacttacaa
     6421 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca
     6481 ctcccaacga agacaagata tccttgatct gtggatctac cacacacaag gctacttccc
     6541 tgattggcag aactacacac cagggccagg gatcagatat ccactgacct ttggatggtg
     6601 ctacaagcta gtaccagttg agcaagagaa ggtagaagaa gccaatgaag gagagaacac
     6661 ccgcttgtta caccctgtga gcctgcatgg gatggatgac ccggagagag aagtattaga
     6721 gtggaggttt gacagccgcc tagcatttca tcacatggcc cgagagctgc atccggactg
     6781 tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
     6841 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
     6901 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
     6961 tagcagcatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     7021 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     7081 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     7141 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     7201 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     7261 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     7321 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     7381 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     7441 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     7501 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     7561 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     7621 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     7681 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     7741 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     7801 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     7861 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     7921 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     7981 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     8041 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     8101 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     8161 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     8221 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     8281 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     8341 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     8401 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     8461 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     8521 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     8581 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     8641 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     8701 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     8761 ccgaaaagtg ccacctgac
//
