LOCUS       sequence-255024-        8927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SP-dCas9 compatible gRNA to be used as a positive control for 
            activation experiments (human 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  JOURNAL   Unpublished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            "Unpublished"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-TTN-Puro
FEATURES             Location/Qualifiers
     source          1..8927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        119..422
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          676..1692
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1616..1638
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1700..1719
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1726..2325
                     /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(1726..1745)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2222..2242
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    2349..2937
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2402..2422)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2820..2831)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3144..3777
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(3906..3922)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3906..3922)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3919..3941)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3930..3946
                     /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(3954..3960,3961..3978,3979..3984))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3999..4020
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4137..4154)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4308..4896)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4388..4407)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5067..5858,5859..5927))
                     /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     5690..5709
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5928..6032)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    6080..6214
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6117..6136)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6171..6190
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             6382..7015
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    7062..7187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7684..7917
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             8102..8146
                     /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    8409..8524
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     promoter        8627..8875
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8627..8647
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8798..8817
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        join(8897..8927,1..45)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttgaattctc
       61 gacctcgaga caaatggcag tattcgtcat tagttcatag cccatatatg gagttccgcg
      121 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      181 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      241 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      301 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      361 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      421 tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc
      481 caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg gggggggggg
      541 gcgcgcgcca ggcggggcng ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc
      601 gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga
      661 agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc
      721 gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg
      781 gacggccctt ctcctccggg ctgtaattag cgcttggttt aatgacggct tgtttctttt
      841 ctgtggctgc gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc
      901 tcggggggtg cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc
      961 ggcggctgtg agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag
     1021 gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc
     1081 tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg
     1141 caaccccccc tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg
     1201 ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg
     1261 gtgccgggcg gggcggggcc gcctcgggcc ggggagggct cgggggaggg gcgcggcggc
     1321 ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa
     1381 tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct gtgcggagcc gaaatctggg
     1441 aggcgccgcc gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag
     1501 gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag
     1561 cctcggggct gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc
     1621 ggcttctggc gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc
     1681 tttttcctac agctcctggg caacgtgctg gttattgtgg ccaccatgac cgagtacaag
     1741 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc
     1801 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg
     1861 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     1921 gtcgcggacg acggcgccgc cgtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     1981 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     2041 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     2101 gccaccgtcg gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     2161 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc
     2221 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     2281 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctaatgtac aagtaaagcg
     2341 tctggaacaa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     2401 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     2461 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     2521 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     2581 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     2641 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     2701 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaagctgacg tcctttccat
     2761 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     2821 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     2881 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcctg
     2941 gaattaattc tgcagtcgag acctagaaaa acatggagca atcacaagta gcaatacagc
     3001 agctaccaat gctgattgtg cctggctaga agcacaagag gaggaggagg tgggttttcc
     3061 agtcacacct caggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca
     3121 ctttttaaaa gaaaagaggg gactggaagg gctaattcac tcccaacgaa gacaagatat
     3181 ccttgatctg tggatctacc acacacaagg ctacttccct gattagcaga actacacacc
     3241 agggccaggg gtcagatatc cactgacctt tggatggtgc tacaagctag taccagttga
     3301 gccagataag gtagaagagg ccaataaagg agagaacacc agcttgttac accctgtgag
     3361 cctgcatggg atggatgacc cggagagaga agtgttagag tggaggtttg acagccgcct
     3421 agcatttcat cacgtggccc gagagctgca tccggagtac ttcaagaact gctgatatcg
     3481 agcttgctac aagggacttt ccgctgggga ctttccaggg aggcgtggcc tgggcgggac
     3541 tggggagtgg cgagccctca gatcctgcat ataagcagct gctttttgcc tgtactgggt
     3601 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     3661 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     3721 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta
     3781 gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga
     3841 gtgagaggcc ttgacattgc tagcgtttta ccgtcgacct ctagctagag cttggcgtaa
     3901 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     3961 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     4021 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4081 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     4141 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4201 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4261 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4321 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4381 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4441 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4501 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4561 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4621 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4681 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4741 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4801 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4861 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4921 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     4981 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     5041 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     5101 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     5161 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     5221 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     5281 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5341 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     5401 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     5461 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     5521 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5581 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5641 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     5701 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5761 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     5821 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     5881 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     5941 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     6001 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     6061 gtcgacggat cgggagatca acttgtttat tgcagcttat aatggttaca aataaagcaa
     6121 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     6181 caaactcatc aatgtatctt atcatgtctg gatcaactgg ataactcaag ctaaccaaaa
     6241 tcatcccaaa cttcccaccc cataccctat taccactgcc aattacctgt ggtttcattt
     6301 actctaaacc tgtgattcct ctgaattatt ttcattttaa agaaattgta tttgttaaat
     6361 atgtactaca aacttagtag ttggaagggc taattcactc ccaaagaaga caagatatcc
     6421 ttgatctgtg gatctaccac acacaaggct acttccctga ttagcagaac tacacaccag
     6481 ggccaggggt cagatatcca ctgacctttg gatggtgcta caagctagta ccagttgagc
     6541 cagataaggt agaagaggcc aataaaggag agaacaccag cttgttacac cctgtgagcc
     6601 tgcatgggat ggatgacccg gagagagaag tgttagagtg gaggtttgac agccgcctag
     6661 catttcatca cgtggcccga gagctgcatc cggagtactt caagaactgc tgatatcgag
     6721 cttgctacaa gggactttcc gctggggact ttccagggag gcgtggcctg ggcgggactg
     6781 gggagtggcg agccctcaga tcctgcatat aagcagctgc tttttgcctg tactgggtct
     6841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
     6901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
     6961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     7021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     7081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     7141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     7201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     7261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     7321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     7381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     7441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     7501 aaagtaagac caccgcacag caagcggccg gccgctgatc ttcagacctg gaggaggaga
     7561 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     7621 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     7681 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     7741 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     7801 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     7861 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     7921 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     7981 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     8041 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     8101 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     8161 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     8221 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     8281 attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga
     8341 agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg gatctcgacg
     8401 gtatcgcctt taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag
     8461 acataatagc aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa
     8521 attttcgggt ttattacagg gacagcagag atccagttta tcattagtga acggatctcg
     8581 acggtatcga tcacgagact agcctcgagc ggccgccccc ttcaccgagg gcctatttcc
     8641 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa
     8701 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
     8761 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
     8821 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgcctt
     8881 ggtgaagtct cctttggttt tagagctaga aatagcaagt taaaata
//
