LOCUS       sequence-254244-        8949 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 8949)
  TITLE     dCas9 activator positive control guideRNAs
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8949)
  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            "Unpublished"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-ACTC1-Puro
FEATURES             Location/Qualifiers
     source          1..8949
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        37..285
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     37..57
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     208..227
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        307..382
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        456..759
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        761..1034
                     /label=chicken beta-actin promoter
     intron          1035..2051
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1975..1997
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2059..2078
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2085..2684
                     /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(2085..2104)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2581..2601
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    2708..3296
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2761..2781)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3179..3190)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3503..4136
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(4265..4281)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4265..4281)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4278..4300)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4289..4305
                     /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(4313..4319,4320..4337,4338..4343))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4358..4379
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4496..4513)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4667..5255)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4747..4766)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5426..6217,6218..6286))
                     /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     6049..6068
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6287..6391)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    6439..6573
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6476..6495)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6530..6549
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             6741..7374
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    7421..7546
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    8043..8276
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             8461..8505
                     /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    8768..8883
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
ORIGIN
        1 tcacgagact agcctcgagc ggccgccccc ttcaccgagg gcctatttcc catgattcct
       61 tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta
      121 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
      181 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
      241 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgtggc gccctgccct
      301 ctgctggttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
      361 aaaagtggca ccgagtcggt gctttttttg aattctcgac ctcgagacaa atggcagtat
      421 tcgtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
      481 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
      541 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
      601 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
      661 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
      721 tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc
      781 tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt
      841 taattatttt gtgcagcgat gggggcgggg ggggggggcg cgcgccaggc ggggcggggc
      901 ggggsgrggg gsggggsggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg
      961 cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa
     1021 gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg
     1081 cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg
     1141 acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc
     1201 tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct
     1261 cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg
     1321 gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg
     1381 ggagcgcggc cgggggcggt gccccgcggt gcgggggggg ctgcgagggg aacaaaggct
     1441 gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc
     1501 aaccccccct gcacccccct ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc
     1561 tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg caggtggggg
     1621 tgccgggcgg ggcggggccg cctcgggccg gggagggctc gggggagggg cgcggcggcc
     1681 cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat
     1741 cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg tgcggagccg aaatctggga
     1801 ggcgccgccg caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg
     1861 aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc
     1921 ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg
     1981 gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat gccttcttct
     2041 ttttcctaca gctcctgggc aacgtgctgg ttattgtggc caccatgacc gagtacaagc
     2101 ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg
     2161 cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg
     2221 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
     2281 tcgcggacga cggcgccgcc gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
     2341 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
     2401 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
     2461 ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
     2521 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc
     2581 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
     2641 aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctaatgtaca agtaaagcgt
     2701 ctggaacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     2761 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     2821 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     2881 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     2941 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     3001 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     3061 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aagctgacgt cctttccatg
     3121 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     3181 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     3241 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcctgg
     3301 aattaattct gcagtcgaga cctagaaaaa catggagcaa tcacaagtag caatacagca
     3361 gctaccaatg ctgattgtgc ctggctagaa gcacaagagg aggaggaggt gggttttcca
     3421 gtcacacctc aggtaccttt aagaccaatg acttacaagg cagctgtaga tcttagccac
     3481 tttttaaaag aaaagagggg actggaaggg ctaattcact cccaacgaag acaagatatc
     3541 cttgatctgt ggatctacca cacacaaggc tacttccctg attagcagaa ctacacacca
     3601 gggccagggg tcagatatcc actgaccttt ggatggtgct acaagctagt accagttgag
     3661 ccagataagg tagaagaggc caataaagga gagaacacca gcttgttaca ccctgtgagc
     3721 ctgcatggga tggatgaccc ggagagagaa gtgttagagt ggaggtttga cagccgccta
     3781 gcatttcatc acgtggcccg agagctgcat ccggagtact tcaagaactg ctgatatcga
     3841 gcttgctaca agggactttc cgctggggac tttccaggga ggcgtggcct gggcgggact
     3901 ggggagtggc gagccctcag atcctgcata taagcagctg ctttttgcct gtactgggtc
     3961 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
     4021 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
     4081 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtag
     4141 tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag
     4201 tgagaggcct tgacattgct agcgttttac cgtcgacctc tagctagagc ttggcgtaat
     4261 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4321 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     4381 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4441 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4501 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4561 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4621 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4681 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4741 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4801 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4861 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4921 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4981 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5041 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5101 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5161 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5221 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5281 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5341 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5401 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5461 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5521 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5581 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5641 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5701 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     5761 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5821 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     5881 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     5941 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6001 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6061 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6121 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6181 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6241 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6301 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6361 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     6421 tcgacggatc gggagatcaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     6481 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     6541 aaactcatca atgtatctta tcatgtctgg atcaactgga taactcaagc taaccaaaat
     6601 catcccaaac ttcccacccc ataccctatt accactgcca attacctgtg gtttcattta
     6661 ctctaaacct gtgattcctc tgaattattt tcattttaaa gaaattgtat ttgttaaata
     6721 tgtactacaa acttagtagt tggaagggct aattcactcc caaagaagac aagatatcct
     6781 tgatctgtgg atctaccaca cacaaggcta cttccctgat tagcagaact acacaccagg
     6841 gccaggggtc agatatccac tgacctttgg atggtgctac aagctagtac cagttgagcc
     6901 agataaggta gaagaggcca ataaaggaga gaacaccagc ttgttacacc ctgtgagcct
     6961 gcatgggatg gatgacccgg agagagaagt gttagagtgg aggtttgaca gccgcctagc
     7021 atttcatcac gtggcccgag agctgcatcc ggagtacttc aagaactgct gatatcgagc
     7081 ttgctacaag ggactttccg ctggggactt tccagggagg cgtggcctgg gcgggactgg
     7141 ggagtggcga gccctcagat cctgcatata agcagctgct ttttgcctgt actgggtctc
     7201 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     7261 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     7321 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
     7381 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg
     7441 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat
     7501 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg
     7561 gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa
     7621 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct
     7681 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac
     7741 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca
     7801 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa
     7861 aagtaagacc accgcacagc aagcggccgg ccgctgatct tcagacctgg aggaggagat
     7921 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
     7981 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
     8041 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     8101 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     8161 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     8221 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     8281 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     8341 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     8401 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     8461 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     8521 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     8581 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     8641 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
     8701 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg
     8761 tatcgccttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
     8821 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa
     8881 ttttcgggtt tattacaggg acagcagaga tccagtttat cattagtgaa cggatctcga
     8941 cggtatcga
//
