LOCUS       sequence-451492-        5890 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ubiquitous expression plasmid, CAG promoter (CMV immediate early 
            enhancer, chicken beta actin promoter), three unique Pax7 gRNAs with
            ribozyme self-cleavage sites, and nuclear split GFP(11) reporter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5890)
  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 5890)
  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-nucGFP11-3xPax7gRNA
FEATURES             Location/Qualifiers
     source          1..5890
                     /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          1814..2830
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2754..2776
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2838..2857
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2968..3015
                     /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             3022..3399
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     ncRNA           3427..3463
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        3464..3493
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     ncRNA           3517..3584
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     ncRNA           3607..3643
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        3644..3673
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     ncRNA           3697..3764
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     ncRNA           3787..3823
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        3824..3853
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     ncRNA           3877..3944
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(4044..4063)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4109..4164
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4163..4182)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4555..4571)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4555..4571)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4568..4590)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4579..4595
                     /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(4603..4609,4610..4627,4628..4633))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4648..4669
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4728..4923
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4774..4909
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4929..5063
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4966..4985)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5020..5039
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5131..5148)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5302..5890)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5382..5401)
                     /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 ggggcgcgcg
     1681 ccaggcgggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc
     1741 gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag
     1801 cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc
     1861 ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga
     1921 cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct
     1981 gtggctgcgt gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc
     2041 ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg
     2101 cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg
     2161 gagcgcggcc gggggcggtg ccccgcggtg cggggggggc tgcgagggga acaaaggctg
     2221 cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca
     2281 accccccctg cacccccctc cccgagttgc tgagcacggc ccggcttcgg gtgcggggct
     2341 ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc aggtgggggt
     2401 gccgggcggg gcggggccgc ctcgggccgg ggagggctcg ggggaggggc gcggcggccc
     2461 ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc cattgccttt tatggtaatc
     2521 gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt gcggagccga aatctgggag
     2581 gcgccgccgc accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga
     2641 aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc
     2701 tcggggctgt ccgcgggggg acggctgcct tcggggggga cggggcaggg cggggttcgg
     2761 cttctggcgt gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt
     2821 tttcctacag ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga
     2881 attgctcgag agttcgaagc cttaagtacg cgtaggcgcg ccttaattaa cgtttaaaca
     2941 tgcatgcaaa tggctagcgc caccatgcgt gaccacatgg tccttcatga gtatgtaaat
     3001 gctgctggga ttacaatggt tatgccagag ccagcgaagt ctgctcccgc cccgaaaaag
     3061 ggctccaaga aggcggtgac taaggcgcag aagaaaggcg gcaagaagcg caagcgcagc
     3121 cgcaaggaga gctattccat ctatgtgtac aaggttctga agcaggtcca ccctgacacc
     3181 ggcatttcgt ccaaggccat gggcatcatg aattcgtttg tgaacgacat tttcgagcgc
     3241 atcgcaggtg aggcttcccg cctggcgcat tacaacaagc gctcgaccat cacctccagg
     3301 gagatccaga cggccgtgcg cctgctgctg cctggggagt tggccaagca cgccgtgtcc
     3361 gagggtacta aggccatcac caagtacacc agcgctaagg atccaccagt cgccaccggt
     3421 cagtgcctga tgagtccgtg aggacgaaac gagtaagctc gtcaacccct accaactggt
     3481 cggggtttga aacccgaatt tgatgcgtag cacacgggcc ggcatggtcc cagcctcctc
     3541 gctggcgccg gctgggcaac atgcttcggc atggcgaatg ggacatcgat acagcgcaaa
     3601 attaggctga tgagtccgtg aggacgaaac gagtaagctc gtcaacccct accaactggt
     3661 cggggtttga aacccctttg tcgcccagga tgccatggcc ggcatggtcc cagcctcctc
     3721 gctggcgccg gctgggcaac atgcttcggc atggcgaatg ggacaagctt acagcgcaaa
     3781 attaggctga tgagtccgtg aggacgaaac gagtaagctc gtcaacccct accaactggt
     3841 cggggtttga aacattcatg tggttggcag gagctgggcc ggcatggtcc cagcctcctc
     3901 gctggcgccg gctgggcaac atgcttcggc atggcgaatg ggactctaga tgagcggccg
     3961 caattcactc ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc
     4021 ctggctcaca aataccactg agatcttttt ccctctgcca aaaattatgg ggacatcatg
     4081 aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg
     4141 tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa
     4201 catcagaatg agtatttggt ttagagtttg gcaacatatg cccatatgct ggctgccatg
     4261 aacaaaggtt ggctataaag aggtcatcag tatatgaaac agccccctgc tgtccattcc
     4321 ttattccata gaaaagcctt gacttgaggt tagatttttt ttatattttg ttttgtgtta
     4381 tttttttctt taacatccct aaaattttcc ttacatgttt tactagccag atttttcctc
     4441 ctctcctgac tactcccagt catagctgtc cctcttctct tatggagatc cctcgacctg
     4501 caccgtcgac cagctggtcg acggtgcacc gtcgaccagc ttggcgtaat catggtcata
     4561 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     4621 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     4681 ctcactgccc gctttccagt cgggaaacct gtcgtgccag cggatccgca tctcaattag
     4741 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4801 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4861 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     4921 aaaaagctaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4981 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     5041 atgtatctta tcatgtctgg atccgctgca ttaatgaatc ggccaacgcg cggggagagg
     5101 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     5161 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     5221 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5281 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5341 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5401 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5461 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5521 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5581 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5641 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5701 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5761 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5821 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5881 aggatctcaa
//
