LOCUS       sequence-388709-        4045 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA targeting upstream region of KCNB2 (site 3418711, control for 
            epigenetic targeting).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4045)
  AUTHORS   Aitken KJ, Jiang JX, Schroder A, Sidler M, Samiei A, Kirwan T, Tolg 
            C, Choufani S, Delgado-Olguin P, Weksberg R,, Bagli DJ.
  TITLE     Persistent changes in KCNB2 expression and CpG methylation is 
            associated with ongoing hypertrophic changes after release of 
            partial outlet obstruction.
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4045)
  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            "Unpublished"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: B52_puro_gRNA_3418711_KCNB2
FEATURES             Location/Qualifiers
     source          1..4045
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        82..330
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        351..426
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        440..688
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        707..782
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        807..1092
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1372..1600
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1618..2217
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1618..1637)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     rep_origin      complement(2296..2884)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2376..2395)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3055..3846,3847..3915))
                     /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     3678..3697
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3916..4020)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtcgcta gctgtacaaa aaagcaggct ttaaaggaac caattcagtc gactggatcc
       61 ggtaccaagg tcgggcagga agagggccta tttcccatga ttccttcata tttgcatata
      121 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
      181 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
      241 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
      301 ttatatatct tgtggaaagg acgaaacacc gcatccccgc cgcccacccc gttttagagc
      361 tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
      421 cggtgctttt tttaagcttg agggcctatt tcccatgatt ccttcatatt tgcatatacg
      481 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt
      541 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
      601 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
      661 atatatcttg tggaaaggac gaaacaccgg gtcttcgaga agacctgttt tagagctaga
      721 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
      781 gctttttttc tagagaattc ctcgagcgtt acataactta cggtaaatgg cccgcctggc
      841 tgaccgccca acgacccccg cccattgacg tcaatagtaa cgccaatagg gactttccat
      901 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
      961 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattgt
     1021 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     1081 gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc
     1141 tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg
     1201 gggggggggg gggggcgcgc gccrggsggg gggggggggg gggggggggg ggggggggsg
     1261 rggsggrgrg gkgsggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg
     1321 gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct
     1381 gcgacgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct
     1441 ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg
     1501 taattagctg agcaagaggt aagggtttaa gggatggttg gttggtgggg tattaatgtt
     1561 taattacctg gagcacctgc ctgaaatcac tttttttcag gttggaccgg tgccaccatg
     1621 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
     1681 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
     1741 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     1801 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     1861 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     1921 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     1981 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc
     2041 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     2101 acctccgcgc cccacaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     2161 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgagta
     2221 cctctctaca tatgacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     2281 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     2341 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     2401 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     2461 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     2521 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     2581 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     2641 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     2701 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     2761 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     2821 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     2881 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     2941 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     3001 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     3061 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     3121 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     3181 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     3241 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     3301 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     3361 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     3421 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     3481 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     3541 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     3601 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     3661 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     3721 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     3781 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     3841 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     3901 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     3961 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     4021 cacatttccc cgaaaagtgc cacct
//
