LOCUS       sequence-451493-        5721 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ubiquitous expression plasmid with CAG promoter (CMV immediate early
            enhancer, chicken beta actin promoter), three unique Pax7 gRNAs with
            ribozyme self-cleavage, three membrane split GFP(11) reporter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5721)
  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 5721)
  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-3xGFP11-mem-3xPax7gRNA
FEATURES             Location/Qualifiers
     source          1..5721
                     /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          1813..2829
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2753..2775
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2837..2856
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2958..2967
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3090..3137
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     CDS             3144..3191
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     CDS             3198..3245
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     ncRNA           3258..3294
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        3295..3324
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     ncRNA           3348..3415
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     ncRNA           3438..3474
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        3475..3504
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     ncRNA           3528..3595
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     ncRNA           3618..3654
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        3655..3684
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     ncRNA           3708..3775
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(3875..3894)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3940..3995
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3994..4013)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4386..4402)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4386..4402)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4399..4421)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4410..4426
                     /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(4434..4440,4441..4458,4459..4464))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4479..4500
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4559..4754
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4605..4740
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4760..4894
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4797..4816)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4851..4870
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4962..4979)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5133..5721)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5213..5232)
                     /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 gggcgcgcgc
     1681 caggcggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg
     1741 ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc
     1801 gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc
     1861 tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac
     1921 ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg
     1981 tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg
     2041 gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc
     2101 ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg
     2161 agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc
     2221 gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa
     2281 ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc
     2341 cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg
     2401 ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc
     2461 cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg
     2521 tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg
     2581 cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa
     2641 atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct
     2701 cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc
     2761 ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt
     2821 ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa
     2881 ttgctcgaga gttcgaagcc ttaagtacgc gtaggcgcgc cttaattaac gtttaaacat
     2941 gcatgcaaat ggctagcgcc accatgggat gcattaaatc taagaggaaa gataacttga
     3001 acgatgacga agctgcaatg gggtgtatta aatccaaaag gaaagataat ttgaatgatg
     3061 atgaagcggg tagtggtgga agtggtggta gggatcatat ggtgctccac gaatacgtga
     3121 acgccgccgg aataactggt gggcgcgatc atatggtgtt acacgaatac gtgaacgcag
     3181 ccggcatcac cggcggacgc gatcatatgg ttttgcacga atacgtgaac gccgcgggaa
     3241 tcactaccgg tcagtgcctg atgagtccgt gaggacgaaa cgagtaagct cgtcaacccc
     3301 taccaactgg tcggggtttg aaacccgaat ttgatgcgta gcacacgggc cggcatggtc
     3361 ccagcctcct cgctggcgcc ggctgggcaa catgcttcgg catggcgaat gggacatcga
     3421 tacagcgcaa aattaggctg atgagtccgt gaggacgaaa cgagtaagct cgtcaacccc
     3481 taccaactgg tcggggtttg aaaccccttt gtcgcccagg atgccatggc cggcatggtc
     3541 ccagcctcct cgctggcgcc ggctgggcaa catgcttcgg catggcgaat gggacaagct
     3601 tacagcgcaa aattaggctg atgagtccgt gaggacgaaa cgagtaagct cgtcaacccc
     3661 taccaactgg tcggggtttg aaacattcat gtggttggca ggagctgggc cggcatggtc
     3721 ccagcctcct cgctggcgcc ggctgggcaa catgcttcgg catggcgaat gggactctag
     3781 atgagcggcc gcaattcact cctcaggtgc aggctgccta tcagaaggtg gtggctggtg
     3841 tggccaatgc cctggctcac aaataccact gagatctttt tccctctgcc aaaaattatg
     3901 gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca
     3961 ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa
     4021 atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat gcccatatgc
     4081 tggctgccat gaacaaaggt tggctataaa gaggtcatca gtatatgaaa cagccccctg
     4141 ctgtccattc cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt
     4201 gttttgtgtt atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca
     4261 gatttttcct cctctcctga ctactcccag tcatagctgt ccctcttctc ttatggagat
     4321 ccctcgacct gcaccgtcga ccagctggtc gacggtgcac cgtcgaccag cttggcgtaa
     4381 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     4441 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     4501 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc
     4561 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     4621 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     4681 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     4741 taggcttttg caaaaagcta acttgtttat tgcagcttat aatggttaca aataaagcaa
     4801 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4861 caaactcatc aatgtatctt atcatgtctg gatccgctgc attaatgaat cggccaacgc
     4921 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     4981 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     5041 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     5101 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     5161 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5221 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5281 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     5341 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     5401 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     5461 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     5521 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     5581 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     5641 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     5701 cgcagaaaaa aaggatctca a
//
