LOCUS       sequence-161546-        9086 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Sp sgSV40 gRNA with ABA-inducible KRAB and mCherry for 
            diametric regulation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9086)
  AUTHORS   Gao Y, Xiong X, Wong S, Charles EJ, Lim WA, Qi LS
  TITLE     Complex transcriptional modulation with orthogonal and inducible 
            dCas9 regulators.
  JOURNAL   Nat Methods. 2016 Oct 24. doi: 10.1038/nmeth.4042.
  PUBMED    27776111
REFERENCE   2  (bases 1 to 9086)
  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: "Nat 
            Methods. 2016 Oct 24. doi: 10.1038/nmeth.4042."
COMMENT     Sequence Label: pSLQ2827 pHR: U6-SpsgSV40 CMV-PYL1-KRAB-IRES-m...
FEATURES             Location/Qualifiers
     source          1..9086
                     /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        3177..3480
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3481..3684
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             4346..4531
                     /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    join(4549..5122,5123..5125,5126..5134)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4716..4733)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4943..4962
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5159..5866
                     /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(5301..5319)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5569..5588
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(5886..5919)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5975..6563
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6028..6048)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6446..6457)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6566..6582)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6567..6583)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             7092..7272
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7334..7922)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7414..7433)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8093..8884,8885..8953))
                     /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     8716..8735
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8954..9058)
                     /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 cacggacttg 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 gttggaaagt ccccaggctc cccagcgttt aagagctatg ctggaaacag catagcaagt
     3001 ttaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttc
     3061 tcgagtacta ggatccatta ggcggccgcg tggataaccg tattaccgcc atgcattagt
     3121 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
     3181 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
     3241 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
     3301 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
     3361 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
     3421 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
     3481 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
     3541 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
     3601 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
     3661 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gctttgcatg
     3721 cggagctacc atgggtgggg gcgcgccaac tcaagacgaa ttcacccaac tctcccaatc
     3781 aatcgccgag ttccacacgt accaactcgg taacggccgt tgctcatctc tcctagctca
     3841 gcgaatccac gcgccgccgg aaacagtatg gtccgtggtg agacgtttcg ataggccaca
     3901 gatttacaaa cacttcatca aaagctgtaa cgtgagtgaa gatttcgaga tgcgagtggg
     3961 atgcacgcgc gacgtgaacg tgataagtgg attaccggcg aatacgtctc gagagagatt
     4021 agatctgttg gacgatgatc ggagagtgac tgggtttagt ataaccggtg gtgaacatag
     4081 gctgaggaat tataaatcgg ttacgacggt tcatagattt gagaaagaag aagaagaaga
     4141 aaggatctgg accgttgttt tggaatctta tgttgttgat gtaccggaag gtaattcgga
     4201 ggaagatacg agattgtttg ctgatacggt tattagattg aatcttcaga aacttgcttc
     4261 gatcactgaa gctatgaacg tgatcactgc aggtactagt ggcggtggcg gagggatgga
     4321 tgctaagtca ctaactgcct ggtcccggac actggtgacc ttcaaggatg tatttgtgga
     4381 cttcaccagg gaggagtgga agctgctgga cactgctcag cagatcgtgt acagaaatgt
     4441 gatgctggag aactataaga acctggtttc cttgggttat cagcttacta agccagatgt
     4501 gatcctccgg ttggagaagg gagaagagcc ctaggtttaa acaattcgcc cctctccctc
     4561 ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta
     4621 tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc
     4681 tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct
     4741 gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt
     4801 agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa
     4861 gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg
     4921 gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg ggctgaagga
     4981 tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac
     5041 gtgtgtttag tcgaggttaa aaaacgtcta ggccccccga accacgggga cgtggttttc
     5101 ctttgaaaaa cacgatgata atatggccac aaccggatcc ggagctacca tgcccggggt
     5161 gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca
     5221 catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc
     5281 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc
     5341 ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc
     5401 cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat
     5461 gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga
     5521 gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca
     5581 gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct
     5641 gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt
     5701 caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat
     5761 caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc
     5821 cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtaagaat tcgtcgaggg
     5881 acctaataac ttcgtatagc atacattata cgaagttata catgtttaag ggttccggtt
     5941 ccactaggta caattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg
     6001 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     6061 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     6121 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     6181 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     6241 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     6301 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     6361 gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg
     6421 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     6481 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
     6541 ctccctttgg gccgcctccc cgcatcgata ccgtcgacct cgatcgagac ctagaaaaac
     6601 atggagcaat cacaagtagc aatacagcag ctaccaatgc tgattgtgcc tggctagaag
     6661 cacaagagga ggaggaggtg ggttttccag tcacacctca ggtaccttta agaccaatga
     6721 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
     6781 taattcactc ccaacgaaga caagatatcc ttgatctgtg gatctaccac acacaaggct
     6841 acttccctga ttggcagaac tacacaccag ggccagggat cagatatcca ctgacctttg
     6901 gatggtgcta caagctagta ccagttgagc aagagaaggt agaagaagcc aatgaaggag
     6961 agaacacccg cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag
     7021 tattagagtg gaggtttgac agccgcctag catttcatca catggcccga gagctgcatc
     7081 cggactgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
     7141 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
     7201 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
     7261 aaatctctag cagcatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     7321 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     7381 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     7441 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     7501 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     7561 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     7621 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     7681 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     7741 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     7801 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     7861 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     7921 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     7981 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     8041 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     8101 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     8161 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     8221 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     8281 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     8341 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     8401 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     8461 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     8521 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     8581 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     8641 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     8701 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     8761 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     8821 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     8881 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     8941 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     9001 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     9061 catttccccg aaaagtgcca cctgac
//
