LOCUS       sequence-97600-e        3226 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses guide RNA used to activate pGL3-Basic-8x-gRNA-eGFP 
            reporter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3226)
  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 3226)
  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: gRNA-eGFP-Reporter
FEATURES             Location/Qualifiers
     source          1..3226
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        659..907
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     659..679
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_RNA        929..1004
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1038..1058)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1038..1056)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1077..1093)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1077..1093)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1090..1112)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1101..1117
                     /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(1125..1131,1132..1149,1150..1155))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1170..1191
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1308..1325)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1479..2067)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1559..1578)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2238..3029,3030..3098))
                     /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     2861..2880
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3099..3203)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccga
      661 gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat
      721 aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa
      781 gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat
      841 gcttatcgta acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg
      901 aaacaccgaa aggtcgagaa actgcaaagt tttagagcta gaaatagcaa gttaaaataa
      961 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tccgcggtgg
     1021 agctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca
     1081 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     1141 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     1201 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     1261 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     1321 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     1381 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     1441 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     1501 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     1561 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     1621 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     1681 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     1741 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     1801 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     1861 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     1921 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     1981 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     2041 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     2101 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     2161 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     2221 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     2281 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     2341 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     2401 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     2461 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     2521 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     2581 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     2641 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     2701 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     2761 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     2821 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     2881 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     2941 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     3001 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     3061 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     3121 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     3181 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccac
//
