LOCUS       sequence-388707-        4044 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sites for cloning two gRNA with puromycin cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4044)
  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  JOURNAL   Unpublished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 4044)
  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            "Unpublished"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_empty_gRNA
FEATURES             Location/Qualifiers
     source          1..4044
                     /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        350..425
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        439..687
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        706..781
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        806..1091
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1371..1599
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1617..2216
                     /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(1617..1636)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     rep_origin      complement(2295..2883)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2375..2394)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3054..3845,3846..3914))
                     /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     3677..3696
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3915..4019)
                     /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 gagacgatta atgcgtctcg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
      421 ggtgcttttt ttaagcttga gggcctattt cccatgattc cttcatattt gcatatacga
      481 tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta
      541 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
      601 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta
      661 tatatcttgt ggaaaggacg aaacaccggg tcttcgagaa gacctgtttt agagctagaa
      721 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
      781 ctttttttct agagaattcc tcgagcgtta cataacttac ggtaaatggc ccgcctggct
      841 gaccgcccaa cgacccccgc ccattgacgt caatagtaac gccaataggg actttccatt
      901 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      961 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattgtg
     1021 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
     1081 ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct
     1141 ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg
     1201 gggggggggg ggggcgcgcg ccrggsgggg sggggggggg gggggggggg ggsggggsgr
     1261 ggsggrgrgg kgcggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg
     1321 cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg
     1381 cgacgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc
     1441 tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt
     1501 aattagctga gcaagaggta agggtttaag ggatggttgg ttggtggggt attaatgttt
     1561 aattacctgg agcacctgcc tgaaatcact ttttttcagg ttggaccggt gccaccatga
     1621 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca
     1681 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc
     1741 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     1801 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg
     1861 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     1921 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
     1981 cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg
     2041 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
     2101 cctccgcgcc ccacaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     2161 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgagtac
     2221 ctctctacat atgacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     2281 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     2341 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     2401 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     2461 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     2521 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     2581 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     2641 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     2701 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     2761 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     2821 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     2881 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     2941 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     3001 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     3061 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     3121 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     3181 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     3241 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     3301 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     3361 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     3421 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     3481 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     3541 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     3601 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     3661 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     3721 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     3781 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     3841 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     3901 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     3961 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     4021 acatttcccc gaaaagtgcc acct
//
