LOCUS       sequence-97599-e        4188 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  eGFP reporter containing 8 copies of a gRNA binding site for 
            light-inducible dCas9 activation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4188)
  AUTHORS   Polstein LR, Gersbach CA
  TITLE     A light-inducible CRISPR-Cas9 system for control of endogenous gene 
            activation.
  JOURNAL   Nat Chem Biol. 2015 Feb 9. doi: 10.1038/nchembio.1753.
  PUBMED    25664691
REFERENCE   2  (bases 1 to 4188)
  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: "Nat Chem 
            Biol. 2015 Feb 9. doi: 10.1038/nchembio.1753."
COMMENT     Sequence Label: pGL3-Basic-8x-gRNA-eGFP
FEATURES             Location/Qualifiers
     source          1..4188
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        201..239
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     236..260
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      290..299
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(296..298,299..301,302..1015)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(296..298,299..301,302..1012)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(341..362)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(602..621)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     949..970
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1151..1272
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1162..1181)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1216..1235
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1520..1537)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1691..2279)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1771..1790)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2450..3241,3242..3310))
                     /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     3073..3092
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3311..3415)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3442..3897
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3529..3548)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3739..3760
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    4028..4076
                     /note="synthetic polyadenylation signal"
     misc_feature    4090..4181
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4130..4149
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggtaccgagc tcttacgcgt gctagcccgg gctcgagaaa actgcaaaag gtcgagaaac
       61 tgcaaaaggt cgagaaactg caaaaggtcg agaaactgca aaaggtcgag aaactgcaaa
      121 aggtcgagaa actgcaaaag gtcgagaaac tgcaaaaggt cgagaaactg caaaaggtcg
      181 agaaactgca aaagagatct ggtaggcgtg tacggtggga ggtctatata agcagagctc
      241 gtttagtgaa ccgtcagatc aagcttggca ttccggtact gttggtaaag ccaccatggt
      301 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
      361 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
      421 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
      481 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
      541 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
      601 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
      661 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
      721 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
      781 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
      841 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
      901 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
      961 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaagctag
     1021 cgtttaaact taagcttggt accgagctcg gatccactag tccagtgtgg tggaattctg
     1081 cagatatcca gcacagtggc ggccgctcga gtctagagtc ggggcggccg gccgcttcga
     1141 gcagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa
     1201 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     1261 aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg
     1321 tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtaaaatcga taaggatccg
     1381 tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat
     1441 gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc
     1501 ggcagcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     1561 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     1621 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     1681 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     1741 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     1801 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     1861 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     1921 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     1981 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     2041 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     2101 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     2161 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     2221 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     2281 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     2341 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     2401 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     2461 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     2521 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     2581 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     2641 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     2701 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     2761 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     2821 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     2881 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     2941 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     3001 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     3061 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     3121 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     3181 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     3241 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     3301 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     3361 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     3421 ttccccgaaa agtgccacct gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3481 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3541 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3601 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3661 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     3721 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     3781 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     3841 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttgcc
     3901 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     3961 tacgccagcc caagctacca tgataagtaa gtaatattaa ggtacgggag gtacttggag
     4021 cggccgcaat aaaatatctt tattttcatt acatctgtgt gttggttttt tgtgtgaatc
     4081 gatagtacta acatacgctc tccatcaaaa caaaacgaaa caaaacaaac tagcaaaata
     4141 ggctgtcccc agtgcaagtg caggtgccag aacatttctc tatcgata
//
