LOCUS       sequence-122972-        3225 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses guide RNA targeting near the stop codon of myogenin in the
            mouse genome..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3225)
  AUTHORS   Gersbach
  TITLE     TMG reporters
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3225)
  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            "Unpublished"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: pZDonor-U6-myogenin stop codon gRNA
FEATURES             Location/Qualifiers
     source          1..3225
                     /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        928..1003
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1037..1057)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1037..1055)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1076..1092)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1076..1092)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1089..1111)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1100..1116
                     /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(1124..1130,1131..1148,1149..1154))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1169..1190
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1307..1324)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1478..2066)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1558..1577)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2237..3028,3029..3097))
                     /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     2860..2879
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3098..3202)
                     /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 aaacaccgca ctcccttacg tccatcggtt ttagagctag aaatagcaag ttaaaataag
      961 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ccgcggtgga
     1021 gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat
     1081 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     1141 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     1201 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     1261 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     1321 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     1381 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     1441 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     1501 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     1561 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     1621 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     1681 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     1741 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     1801 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     1861 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     1921 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     1981 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     2041 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     2101 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     2161 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     2221 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     2281 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     2341 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     2401 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     2461 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     2521 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     2581 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     2641 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     2701 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     2761 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     2821 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     2881 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     2941 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     3001 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     3061 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     3121 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     3181 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccac
//
