LOCUS       sequence-369410-        2146 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used to synthesize gRNA targeting exon 2 of the gsc2 gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2146)
  AUTHORS   Spikol ED, Cheng J, Macurak M, Subedi A, Halpern ME
  TITLE     Genetically defined nucleus incertus neurons differ in connectivity 
            and function.
  JOURNAL   Elife. 2024 May 31;12:RP89516. doi: 10.7554/eLife.89516.
  PUBMED    38819436
REFERENCE   2  (bases 1 to 2146)
  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: "Elife."; 
            date: "2024-05-31"; volume: "12:RP89516. doi"; pages: " 
            10.7554/eLife.89516"
COMMENT     Sequence Label: pDR274-gsc2-sgRNA
FEATURES             Location/Qualifiers
     source          1..2146
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        76..94
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        115..190
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(372..389)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(372..388)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      429..1016
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     917..936
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1028..1055)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(1079..1894)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCLKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1802..1821
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1895..1997)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(1998..2029)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     primer_bind     2077..2093
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2077..2093
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 cgtaagatca cgggtcgcag cacagctcgc ggtccagtag tgatcgacac tgctcgatcc
       61 gctcgcaccg ctagctaata cgactcacta taggtcaccg caccatcttc acaggtttta
      121 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
      181 gagtcggtgc ttttaaaaag cttggatcga cgagagcagc gcgactggat ctgtcgcccg
      241 tctcaaacgc aaccctccgg cggtcgcata tcattcagga cgagcctcag actccagcgt
      301 aactggactg caatcaactc actggctcac cttccggtcc acgatcagct agaatcaagc
      361 tgactagata aactggccgt cgttttacac gggtgggcct ttcttcggta gaaaatcaaa
      421 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
      481 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaggtaa
      541 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
      601 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
      661 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
      721 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
      781 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
      841 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
      901 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
      961 tctgacttga gcatcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     1021 ccagcaacgc agaaaggccc acccgaaggt gagccaggtg attacattta ggtcctcatt
     1081 agaaaaactc atcgagcatc aagtgaaact gcaatttatt catatcagga ttatcaatac
     1141 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
     1201 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
     1261 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
     1321 aatccggtga gaatggcaag agtttatgca tttctttcca gacttgttca acaggccagc
     1381 cattacgctc gtcatcaaaa tcactcgcac caaccaaacc gttattcatt cgtgattgcg
     1441 cctgagcgag acgaaatacg cgatcgccgt taaaaggaca attacaaaca ggaatcgaat
     1501 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     1561 cttctaatac ctggaatgct gttttccctg ggatcgcagt ggtgagtaac catgcatcat
     1621 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     1681 gcctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     1741 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
     1801 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggct
     1861 tcaagcaaga cgtttcccgt tgaatatggc tcattttagc ttccttagct cctgaaaatc
     1921 tcgataactc aaaaaatacg cccggtagtg atcttatttc attatggtga aagttggaac
     1981 ctcttacgtg ccgatcaagt caaaagcctc cggtcggagg cttttgactt tctgctatgg
     2041 aggtcaggta tgatttaaat ggtcagtatt gagcctcagg aaacagctat gacatcaagc
     2101 tgactagata atctagctga tcgtggaccg atcatacgta taatgc
//
