LOCUS       sequence-255029-        8927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SP-dCas9 compatible gRNA to be used as a positive control for 
            activation experiments (mouse cells).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8927)
  TITLE     dCas9 activator positive control guideRNAs
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8927)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pSB700-mouse-ACTC1-Puro
FEATURES             Location/Qualifiers
     source          1..8927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          126..1142
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1066..1088
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1150..1169
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1176..1775
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1176..1195)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1672..1692
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    1799..2387
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1852..1872)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2270..2281)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2594..3227
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(3356..3372)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3356..3372)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3369..3391)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3380..3396
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3404..3410,3411..3428,3429..3434))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3449..3470
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3587..3604)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3758..4346)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3838..3857)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4517..5308,5309..5377))
                     /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     5140..5159
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5378..5482)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    5530..5664
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5567..5586)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5621..5640
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             5832..6465
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    6512..6637
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7134..7367
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7552..7596
                     /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    7859..7974
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     promoter        8077..8325
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8077..8097
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8248..8267
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8347..8422
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        8496..8799
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa
       61 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc
      121 gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc
      181 gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt
      241 ctcctccggg ctgtaattag cgcttggttt aatgacggct tgtttctttt ctgtggctgc
      301 gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg
      361 cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg
      421 agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg
      481 ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg
      541 gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc
      601 tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg
      661 ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg
      721 gggcggggcc gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg
      781 ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga
      841 gggcgcaggg acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc
      901 gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg
      961 gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct
     1021 gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc
     1081 gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac
     1141 agctcctggg caacgtgctg gttattgtgg ccaccatgac cgagtacaag cccacggtgc
     1201 gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg
     1261 actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc
     1321 tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg
     1381 acggcgccgc cgtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg
     1441 ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga
     1501 tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg
     1561 gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg
     1621 aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc
     1681 ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc
     1741 gcacctggtg catgacccgc aagcccggtg cctaatgtac aagtaaagcg tctggaacaa
     1801 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     1861 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     1921 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     1981 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     2041 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     2101 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     2161 gggcactgac aattccgtgg tgttgtcggg gaagctgacg tcctttccat ggctgctcgc
     2221 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     2281 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     2341 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcctg gaattaattc
     2401 tgcagtcgag acctagaaaa acatggagca atcacaagta gcaatacagc agctaccaat
     2461 gctgattgtg cctggctaga agcacaagag gaggaggagg tgggttttcc agtcacacct
     2521 caggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa
     2581 gaaaagaggg gactggaagg gctaattcac tcccaacgaa gacaagatat ccttgatctg
     2641 tggatctacc acacacaagg ctacttccct gattagcaga actacacacc agggccaggg
     2701 gtcagatatc cactgacctt tggatggtgc tacaagctag taccagttga gccagataag
     2761 gtagaagagg ccaataaagg agagaacacc agcttgttac accctgtgag cctgcatggg
     2821 atggatgacc cggagagaga agtgttagag tggaggtttg acagccgcct agcatttcat
     2881 cacgtggccc gagagctgca tccggagtac ttcaagaact gctgatatcg agcttgctac
     2941 aagggacttt ccgctgggga ctttccaggg aggcgtggcc tgggcgggac tggggagtgg
     3001 cgagccctca gatcctgcat ataagcagct gctttttgcc tgtactgggt ctctctggtt
     3061 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
     3121 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
     3181 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta gtagttcatg
     3241 tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggcc
     3301 ttgacattgc tagcgtttta ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     3361 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     3421 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     3481 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     3541 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     3601 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     3661 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     3721 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     3781 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     3841 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     3901 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     3961 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4021 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4081 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     4141 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     4201 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     4261 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     4321 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     4381 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     4441 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     4501 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     4561 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     4621 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     4681 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     4741 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     4801 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     4861 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     4921 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     4981 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     5041 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     5101 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     5161 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     5221 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     5281 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     5341 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     5401 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     5461 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
     5521 cgggagatca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     5581 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     5641 aatgtatctt atcatgtctg gatcaactgg ataactcaag ctaaccaaaa tcatcccaaa
     5701 cttcccaccc cataccctat taccactgcc aattacctgt ggtttcattt actctaaacc
     5761 tgtgattcct ctgaattatt ttcattttaa agaaattgta tttgttaaat atgtactaca
     5821 aacttagtag ttggaagggc taattcactc ccaaagaaga caagatatcc ttgatctgtg
     5881 gatctaccac acacaaggct acttccctga ttagcagaac tacacaccag ggccaggggt
     5941 cagatatcca ctgacctttg gatggtgcta caagctagta ccagttgagc cagataaggt
     6001 agaagaggcc aataaaggag agaacaccag cttgttacac cctgtgagcc tgcatgggat
     6061 ggatgacccg gagagagaag tgttagagtg gaggtttgac agccgcctag catttcatca
     6121 cgtggcccga gagctgcatc cggagtactt caagaactgc tgatatcgag cttgctacaa
     6181 gggactttcc gctggggact ttccagggag gcgtggcctg ggcgggactg gggagtggcg
     6241 agccctcaga tcctgcatat aagcagctgc tttttgcctg tactgggtct ctctggttag
     6301 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     6361 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     6421 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag
     6481 ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga
     6541 agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag
     6601 cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag
     6661 atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca
     6721 tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac
     6781 atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga
     6841 agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga
     6901 gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac
     6961 caccgcacag caagcggccg gccgctgatc ttcagacctg gaggaggaga tatgagggac
     7021 aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca
     7081 cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct
     7141 ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg
     7201 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg
     7261 gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag
     7321 gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt
     7381 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa
     7441 tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat
     7501 tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa
     7561 caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat
     7621 tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata
     7681 gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt
     7741 cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt
     7801 ggagagagag acagagacag atccattcga ttagtgaacg gatctcgacg gtatcgcctt
     7861 taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc
     7921 aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt
     7981 ttattacagg gacagcagag atccagttta tcattagtga acggatctcg acggtatcga
     8041 tcacgagact agcctcgagc ggccgccccc ttcaccgagg gcctatttcc catgattcct
     8101 tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta
     8161 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     8221 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     8281 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgctcc cagaccatgt
     8341 aaggaagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
     8401 aaaagtggca ccgagtcggt gctttttttg aattctcgac ctcgagacaa atggcagtat
     8461 tcgtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
     8521 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
     8581 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     8641 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
     8701 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
     8761 tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc
     8821 tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt
     8881 taattatttt gtgcagcgat gggggcgggg ggggggggcg cgcgcca
//
