LOCUS       sequence-245789-        8952 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 8952)
  TITLE     dCas9 activator positive control guideRNAs
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8952)
  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            "Unpublished"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..8952
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /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    2028..2143
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     promoter        2246..2494
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2246..2266
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2417..2436
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2516..2591
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        2665..2968
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2970..3244
                     /label=chicken beta-actin promoter
     intron          3246..4263
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4187..4209
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4271..4290
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             4297..4896
                     /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(4297..4316)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4793..4813
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    4920..5508
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4973..4993)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5391..5402)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5715..6348
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6477..6493)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6477..6493)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6490..6512)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6501..6517
                     /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(6525..6531,6532..6549,6550..6555))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6570..6591
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6708..6725)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6879..7467)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6959..6978)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7638..8429,8430..8498))
                     /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     8261..8280
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8499..8603)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8651..8785
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8688..8707)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8742..8761
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag
     2041 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     2101 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     2161 acagcagaga tccagtttat cattagtgaa cggatctcga cggtatcgat cacgagacta
     2221 gcctcgagcg gccgccccct tcaccgaggg cctatttccc atgattcctt catatttgca
     2281 tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat
     2341 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
     2401 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
     2461 ggctttatat atcttgtgga aaggacgaaa caccgccttg gtgaagtctc ctttggtttt
     2521 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac
     2581 cgagtcggtg ctttttttga attctcgacc tcgagacaaa tggcagtatt cgtcattagt
     2641 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     2701 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     2761 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     2821 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     2881 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     2941 ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct
     3001 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
     3061 tgcagcgatg ggggcggggg ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg
     3121 gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
     3181 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
     3241 ggcggggagt cgctgcgacg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc
     3301 cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct
     3361 tctcctccgg gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg
     3421 cgtgaaagcc ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggggt
     3481 gcgtgcgtgt gtgtgtgcgt ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt
     3541 gagcgctgcg ggcgcggcgc ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg
     3601 gccgggggcg gtgccccgcg gtgcgggggg ggctgcgagg ggaacaaagg ctgcgtgcgg
     3661 ggtgtgtgcg tgggggggtg agcagggggt gtgggcgcgt cggtcgggct gcaacccccc
     3721 ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtacg
     3781 gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc
     3841 ggggcggggc cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg cccccggagc
     3901 gccggcggct gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag
     3961 agggcgcagg gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc
     4021 cgcaccccct ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc
     4081 ggggagggcc ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca gcctcggggc
     4141 tgtccgcggg gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg
     4201 cgtgtgaccg gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta
     4261 cagctcctgg gcaacgtgct ggttattgtg gccaccatga ccgagtacaa gcccacggtg
     4321 cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc
     4381 gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag
     4441 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
     4501 gacggcgccg ccgtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
     4561 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
     4621 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
     4681 ggagtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
     4741 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
     4801 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
     4861 cgcacctggt gcatgacccg caagcccggt gcctaatgta caagtaaagc gtctggaaca
     4921 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     4981 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
     5041 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt
     5101 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
     5161 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta
     5221 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt
     5281 tgggcactga caattccgtg gtgttgtcgg ggaagctgac gtcctttcca tggctgctcg
     5341 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
     5401 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
     5461 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcct ggaattaatt
     5521 ctgcagtcga gacctagaaa aacatggagc aatcacaagt agcaatacag cagctaccaa
     5581 tgctgattgt gcctggctag aagcacaaga ggaggaggag gtgggttttc cagtcacacc
     5641 tcaggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa
     5701 agaaaagagg ggactggaag ggctaattca ctcccaacga agacaagata tccttgatct
     5761 gtggatctac cacacacaag gctacttccc tgattagcag aactacacac cagggccagg
     5821 ggtcagatat ccactgacct ttggatggtg ctacaagcta gtaccagttg agccagataa
     5881 ggtagaagag gccaataaag gagagaacac cagcttgtta caccctgtga gcctgcatgg
     5941 gatggatgac ccggagagag aagtgttaga gtggaggttt gacagccgcc tagcatttca
     6001 tcacgtggcc cgagagctgc atccggagta cttcaagaac tgctgatatc gagcttgcta
     6061 caagggactt tccgctgggg actttccagg gaggcgtggc ctgggcggga ctggggagtg
     6121 gcgagccctc agatcctgca tataagcagc tgctttttgc ctgtactggg tctctctggt
     6181 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     6241 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     6301 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat
     6361 gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag agtgagaggc
     6421 cttgacattg ctagcgtttt accgtcgacc tctagctaga gcttggcgta atcatggtca
     6481 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     6541 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     6601 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     6661 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     6721 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     6781 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     6841 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     6901 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     6961 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     7021 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     7081 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     7141 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     7201 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     7261 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     7321 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     7381 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     7441 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     7501 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     7561 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     7621 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     7681 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     7741 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     7801 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     7861 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     7921 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     7981 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     8041 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     8101 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     8161 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     8221 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     8281 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     8341 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     8401 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     8461 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     8521 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     8581 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     8641 tcgggagatc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     8701 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     8761 caatgtatct tatcatgtct ggatcaactg gataactcaa gctaaccaaa atcatcccaa
     8821 acttcccacc ccatacccta ttaccactgc caattacctg tggtttcatt tactctaaac
     8881 ctgtgattcc tctgaattat tttcatttta aagaaattgt atttgttaaa tatgtactac
     8941 aaacttagta gt
//
