LOCUS       sequence-161100-        7656 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Sp sgCD95-2 gRNA with an EGFP fluorescent marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7656)
  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 7656)
  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: pSLQ1852-3 pHR: U6-SpsgCD95-2 CMV-EGFP
FEATURES             Location/Qualifiers
     source          1..7656
                     /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        3176..3479
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3480..3683
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             join(3717..3719,3720..3722,3723..4436)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3762..3783)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4023..4042)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4370..4391
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(4456..4489)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4545..5133
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4598..4618)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5016..5027)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5136..5152)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5137..5153)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5662..5842
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(5904..6492)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5984..6003)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6663..7454,7455..7523))
                     /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     7286..7305
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7524..7628)
                     /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 gttggtggca tgctcacttc aggtggttta agagctatgc tggaaacagc atagcaagtt
     3001 taaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttct
     3061 cgagtactag gatccattag gcggccgcgt ggataaccgt attaccgcca tgcattagtt
     3121 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     3181 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     3241 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     3301 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
     3361 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     3421 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
     3481 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
     3541 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     3601 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
     3661 gggaggtcta tataagcaga gctggtttag tgaaccgtca gatccgctag cccaccatgg
     3721 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     3781 acgtaaacgg ccacaagttc agcgtgtctg gcgagggcga gggcgatgcc acctacggca
     3841 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     3901 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     3961 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     4021 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     4081 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     4141 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     4201 tcaaggcgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     4261 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     4321 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     4381 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaagaat
     4441 tcgtcgaggg acctaataac ttcgtatagc atacattata cgaagttata catgtttaag
     4501 ggttccggtt ccactaggta caattcgata tcaagcttat cgataatcaa cctctggatt
     4561 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     4621 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     4681 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     4741 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     4801 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     4861 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     4921 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct
     4981 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     5041 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     5101 cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtcgacct cgatcgagac
     5161 ctagaaaaac atggagcaat cacaagtagc aatacagcag ctaccaatgc tgattgtgcc
     5221 tggctagaag cacaagagga ggaggaggtg ggttttccag tcacacctca ggtaccttta
     5281 agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga
     5341 ctggaagggc taattcactc ccaacgaaga caagatatcc ttgatctgtg gatctaccac
     5401 acacaaggct acttccctga ttggcagaac tacacaccag ggccagggat cagatatcca
     5461 ctgacctttg gatggtgcta caagctagta ccagttgagc aagagaaggt agaagaagcc
     5521 aatgaaggag agaacacccg cttgttacac cctgtgagcc tgcatgggat ggatgacccg
     5581 gagagagaag tattagagtg gaggtttgac agccgcctag catttcatca catggcccga
     5641 gagctgcatc cggactgtac tgggtctctc tggttagacc agatctgagc ctgggagctc
     5701 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa
     5761 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag
     5821 tcagtgtgga aaatctctag cagcatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     5881 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5941 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     6001 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     6061 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     6121 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     6181 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     6241 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     6301 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     6361 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     6421 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6481 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6541 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6601 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6661 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6721 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6781 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     6841 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6901 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6961 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     7021 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     7081 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     7141 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     7201 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     7261 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     7321 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     7381 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     7441 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     7501 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     7561 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     7621 gttccgcgca catttccccg aaaagtgcca cctgac
//
