LOCUS       sequence-451497-        6238 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ubiquitous expression plasmid, CAG promoter (CMV immediate early 
            enhancer and chicken beta actin promoter), three unique Pax7 gRNAs 
            with ribozyme self-cleavage sites, and membrane RFP reporter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6238)
  AUTHORS   Kim M, Hutchins EJ
  TITLE     CRISPR-Cas13d as a molecular tool to achieve targeted gene 
            expression knockdown in chick embryos.
  JOURNAL   Dev Biol. 2024 Nov 30:S0012-1606(24)00265-3. doi: 
            10.1016/j.ydbio.2024.11.013.
  PUBMED    39622311
REFERENCE   2  (bases 1 to 6238)
  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: "Dev Biol. 
            2024 Nov 30:S0012-1606(24)00265-3. doi: 
            10.1016/j.ydbio.2024.11.013."
COMMENT     Sequence Label: pCAG-memRFP-3xPax7gRNA
FEATURES             Location/Qualifiers
     source          1..6238
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        1167..1546
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          1811..2827
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2751..2773
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2835..2854
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2956..2965
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3088..3762
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     ncRNA           3775..3811
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        3812..3841
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     ncRNA           3865..3932
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     ncRNA           3955..3991
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        3992..4021
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     ncRNA           4045..4112
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     ncRNA           4135..4171
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        4172..4201
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     ncRNA           4225..4292
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(4392..4411)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4457..4512
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4511..4530)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4903..4919)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4903..4919)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4916..4938)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4927..4943
                     /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(4951..4957,4958..4975,4976..4981))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4996..5017
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5076..5271
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5122..5257
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5277..5411
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5314..5333)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5368..5387
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5479..5496)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5650..6238)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5730..5749)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgggtcgaca ttgattattg actagttatt aatagtaatc
     1201 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     1261 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     1321 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     1381 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     1441 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     1501 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc
     1561 cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt
     1621 tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggggg gcgcgcgcca
     1681 ggcggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct
     1741 ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc
     1801 gcggcgggcg ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc
     1861 gcgccgcccg ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg
     1921 cccttctcct ccgggctgta attagcgctt ggtttaatga cggcttgttt cttttctgtg
     1981 gctgcgtgaa agccttgagg ggctccggga gggccctttg tgcgggggga gcggctcggg
     2041 gggtgcgtgc gtgtgtgtgt gcgtggggag cgccgcgtgc ggctccgcgc tgcccggcgg
     2101 ctgtgagcgc tgcgggcgcg gcgcggggct ttgtgcgctc cgcagtgtgc gcgaggggag
     2161 cgcggccggg ggcggtgccc cgcggtgcgg ggggggctgc gaggggaaca aaggctgcgt
     2221 gcggggtgtg tgcgtggggg ggtgagcagg gggtgtgggc gcgtcggtcg ggctgcaacc
     2281 ccccctgcac ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg
     2341 tacggggcgt ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc
     2401 gggcggggcg gggccgcctc gggccgggga gggctcgggg gaggggcgcg gcggcccccg
     2461 gagcgccggc ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg
     2521 cgagagggcg cagggacttc ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg
     2581 ccgccgcacc ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat
     2641 gggcggggag ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg
     2701 gggctgtccg cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt
     2761 ctggcgtgtg accggcggct ctagagcctc tgctaaccat gttcatgcct tcttcttttt
     2821 cctacagctc ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaagaatt
     2881 gctcgagagt tcgaagcctt aagtacgcgt aggcgcgcct taattaacgt ttaaacatgc
     2941 atgcaaatgg ctagcgccac catgggatgc attaaatcta agaggaaaga taacttgaac
     3001 gatgacgaag ctgcaatggg gtgtattaaa tccaaaagga aagataattt gaatgatgat
     3061 gaagcgggct ccggcggctc tggcggcatg gcaagcagcg aagatgttat taaagaattt
     3121 atgcggttta aagtccgaat ggaaggatca gtaaatggac atgaatttga aattgaagga
     3181 gaaggtgaag gtcgaccata tgaaggaacg caaactgcta aactcaaagt cacgaaaggt
     3241 ggtccattgc cgtttgcttg ggatatactc tctccccaat ttcaatatgg atcaaaagca
     3301 tatgttaaac atcctgcaga tattcctgat tatctgaaac tcagctttcc agaaggcttt
     3361 aaatgggaac gggtaatgaa ttttgaagat ggtggggttg ttacagtcac ccaagattct
     3421 agtcttcaag atggggaatt tatatataaa gtcaaattgc ggggcacaaa ttttccaagc
     3481 gatgggccag tcatgcaaaa gaaaacaatg ggatgggaag catctacaga aaggatgtat
     3541 ccagaagatg gagctctcaa aggggaaatt aaaatgagat tgaaactcaa agatggcggg
     3601 cattatgatg cagaagtgaa aacgacttat atggcgaaga aacccgttca gctgcctggg
     3661 gcttataaaa cagatattaa actcgatatt actagccata atgaagatta cacaatagtc
     3721 gagcaatatg aacgggcaga agggcggcat tcaacaggag ctaccggtca gtgcctgatg
     3781 agtccgtgag gacgaaacga gtaagctcgt caacccctac caactggtcg gggtttgaaa
     3841 cccgaatttg atgcgtagca cacgggccgg catggtccca gcctcctcgc tggcgccggc
     3901 tgggcaacat gcttcggcat ggcgaatggg acatcgatac agcgcaaaat taggctgatg
     3961 agtccgtgag gacgaaacga gtaagctcgt caacccctac caactggtcg gggtttgaaa
     4021 cccctttgtc gcccaggatg ccatggccgg catggtccca gcctcctcgc tggcgccggc
     4081 tgggcaacat gcttcggcat ggcgaatggg acaagcttac agcgcaaaat taggctgatg
     4141 agtccgtgag gacgaaacga gtaagctcgt caacccctac caactggtcg gggtttgaaa
     4201 cattcatgtg gttggcagga gctgggccgg catggtccca gcctcctcgc tggcgccggc
     4261 tgggcaacat gcttcggcat ggcgaatggg actctagatg agcggccgca attcactcct
     4321 caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa
     4381 taccactgag atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag
     4441 catctgactt ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt
     4501 ttgtgtctct cactcggaag gacatatggg agggcaaatc atttaaaaca tcagaatgag
     4561 tatttggttt agagtttggc aacatatgcc catatgctgg ctgccatgaa caaaggttgg
     4621 ctataaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga
     4681 aaagccttga cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta
     4741 acatccctaa aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta
     4801 ctcccagtca tagctgtccc tcttctctta tggagatccc tcgacctgca ccgtcgacca
     4861 gctggtcgac ggtgcaccgt cgaccagctt ggcgtaatca tggtcatagc tgtttcctgt
     4921 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     4981 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     5041 tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc tcaattagtc agcaaccata
     5101 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     5161 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     5221 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctaact
     5281 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     5341 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     5401 atgtctggat ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     5461 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     5521 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     5581 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     5641 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     5701 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     5761 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     5821 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     5881 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     5941 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     6001 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     6061 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     6121 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     6181 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaa
//
