LOCUS       sequence-79087-e        4134 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA expression empty vector (mammalian cells).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4134)
  AUTHORS   Farzadfard F, Perli SD, Lu TK
  TITLE     Tunable and Multifunctional Eukaryotic Transcription Factors Based 
            on CRISPR/Cas.
  JOURNAL   ACS Synth Biol. 2013 Sep 11.
  PUBMED    23977949
REFERENCE   2  (bases 1 to 4134)
  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: "ACS Synth 
            Biol. 2013 Sep 11."
COMMENT     Sequence Label: pU6_gRNA_handle_U6t
FEATURES             Location/Qualifiers
     source          1..4134
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        132..380
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     132..152
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     303..322
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        382..457
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(469..493)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        518..1017
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(553..576)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     950..969
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      1035..1044
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1041..1043,1044..1046,1047..1760)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(1086..1107)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    1927..2048
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1938..1957)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1992..2011
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2279..2296)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2450..3035)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2530..2549)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3206..3997,3998..4066))
                     /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     3829..3848
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(4067..4134,1..37))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
       61 cttgtacaaa aaagcaggct ttaaaggaac caattcagtc gactggatcc ggtaccaagg
      121 tcgggcagga agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
      181 ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata
      241 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
      301 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
      361 tgtggaaagg acggagctcc ggttttagag ctagaaatag caagttaaaa taaggctagt
      421 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttctagac ccagctttct
      481 tgtacaaagt tggcattaga cgtcgaggaa ttctaccggg taggggaggc gcttttccca
      541 aggcagtctg gagcatgcgc tttagcagcc ccgctgggca cttggcgcta cacaagtggc
      601 ctctggcctc gcacacattc cacatccacc ggtaggcgcc aaccggctcc gttctttggt
      661 ggccccttcg cgccaccttc tactcctccc ctagtcagga agttcccccc cgccccgcag
      721 ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc tcactagtct cgtgcagatg
      781 gacagcaccg ctgagcaatg gaagcgggta ggcctttggg gcagcggcca atagcagctt
      841 tgctccttcg ctttctgggc tcagaggctg ggaaggggtg ggtccggggg cgggctcagg
      901 ggcgggctca ggggcggggc gggcgcccga aggtcctccg gaggcccggc attctgcacg
      961 cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct catctccggg cctttcgacc
     1021 tgcatcccaa gcttgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc
     1081 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg aggggcgagg
     1141 gcgagggcga tgccaccaac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     1201 tgcccgtgcc ctggcccacc ctcgtgacca ccctgagcca cggcgtgcag tgcttcgccc
     1261 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     1321 tccaggagcg caccatcttc ttcaaggacg acggcaccta caagacccgc gccgaggtga
     1381 agttcgaggg cgacacccta gtgaaccgca tcgagctgaa gggcgtcgac ttcaaggagg
     1441 acggcaacat cctggggcac aagctggagt acaacttcaa cagccacaac atctatatca
     1501 tggccgtcaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacgtggagg
     1561 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     1621 tgctgctgcc cgacagccac tacctgagca cccagtccgt gctgagcaaa gaccccaacg
     1681 agaagcgcga tcacatggtc ctgctggagt tccgcaccgc cgccgggatc actctcggca
     1741 tggacgagct gtacaagtaa gtaccactag taccggtaga tatcagccat ggcttcccgc
     1801 cggcggtggc ggcgcaggat gatggcacgc tgcccatgtc ttgtgcccag gagagcggga
     1861 tggaccgtca ccctgcagcc tgtgcttctg ctaggatcaa tgtgtaggct agtcgagcag
     1921 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     1981 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     2041 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gagatgtggg
     2101 aggtttttta aagcaagtaa aacctctaca aatgtggtaa aatccgataa ggatcgatcc
     2161 gggctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcctgcatt
     2221 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     2281 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     2341 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     2401 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     2461 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     2521 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     2581 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     2641 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     2701 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     2761 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     2821 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     2881 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     2941 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtttt tttgtttgca
     3001 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     3061 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     3121 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     3181 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     3241 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     3301 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     3361 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     3421 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     3481 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     3541 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     3601 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     3661 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     3721 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     3781 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     3841 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     3901 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     3961 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     4021 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     4081 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttg
//
