LOCUS       sequence-451496-        5888 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ubiquitous expression plasmid, CAG promoter (CMV immediate early 
            enhancer and chicken beta actin promoter), three Control 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 5888)
  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 5888)
  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-nucGFP11-3xControlgRNA
FEATURES             Location/Qualifiers
     source          1..5888
                     /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          1812..2828
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2752..2774
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2836..2855
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2966..3013
                     /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             3020..3397
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     ncRNA           3425..3461
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        3462..3491
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     ncRNA           3515..3582
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     ncRNA           3605..3641
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        3642..3671
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     ncRNA           3695..3762
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     ncRNA           3785..3821
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        3822..3851
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     ncRNA           3875..3942
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(4042..4061)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4107..4162
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4161..4180)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4553..4569)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4553..4569)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4566..4588)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4577..4593
                     /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(4601..4607,4608..4625,4626..4631))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4646..4667
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4726..4921
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4772..4907
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4927..5061
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4964..4983)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5018..5037
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5129..5146)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5300..5888)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5380..5399)
                     /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 ggcgcgcgcc
     1681 aggcggggcg gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc
     1741 tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg
     1801 cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct
     1861 cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg
     1921 gcccttctcc tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt
     1981 ggctgcgtga aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg
     2041 ggggtgcgtg cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg
     2101 gctgtgagcg ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga
     2161 gcgcggccgg gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg
     2221 tgcggggtgt gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac
     2281 cccccctgca cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc
     2341 gtacggggcg tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc
     2401 cgggcggggc ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc
     2461 ggagcgccgg cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt
     2521 gcgagagggc gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc
     2581 gccgccgcac cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa
     2641 tgggcgggga gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc
     2701 ggggctgtcc gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct
     2761 tctggcgtgt gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt
     2821 tcctacagct cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat
     2881 tgctcgagag ttcgaagcct taagtacgcg taggcgcgcc ttaattaacg tttaaacatg
     2941 catgcaaatg gctagcgcca ccatgcgtga ccacatggtc cttcatgagt atgtaaatgc
     3001 tgctgggatt acaatggtta tgccagagcc agcgaagtct gctcccgccc cgaaaaaggg
     3061 ctccaagaag gcggtgacta aggcgcagaa gaaaggcggc aagaagcgca agcgcagccg
     3121 caaggagagc tattccatct atgtgtacaa ggttctgaag caggtccacc ctgacaccgg
     3181 catttcgtcc aaggccatgg gcatcatgaa ttcgtttgtg aacgacattt tcgagcgcat
     3241 cgcaggtgag gcttcccgcc tggcgcatta caacaagcgc tcgaccatca cctccaggga
     3301 gatccagacg gccgtgcgcc tgctgctgcc tggggagttg gccaagcacg ccgtgtccga
     3361 gggtactaag gccatcacca agtacaccag cgctaaggat ccaccagtcg ccaccggtca
     3421 gtgcctgatg agtccgtgag gacgaaacga gtaagctcgt caacccctac caactggtcg
     3481 gggtttgaaa cctcttacct cagttacaat ttatggccgg catggtccca gcctcctcgc
     3541 tggcgccggc tgggcaacat gcttcggcat ggcgaatggg acatcgatac agcgcaaaat
     3601 taggctgatg agtccgtgag gacgaaacga gtaagctcgt caacccctac caactggtcg
     3661 gggtttgaaa cctcttacct cagttacaat ttatggccgg catggtccca gcctcctcgc
     3721 tggcgccggc tgggcaacat gcttcggcat ggcgaatggg acaagcttac agcgcaaaat
     3781 taggctgatg agtccgtgag gacgaaacga gtaagctcgt caacccctac caactggtcg
     3841 gggtttgaaa cctcttacct cagttacaat ttatggccgg catggtccca gcctcctcgc
     3901 tggcgccggc tgggcaacat gcttcggcat ggcgaatggg actctagatg agcggccgca
     3961 attcactcct caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct
     4021 ggctcacaaa taccactgag atctttttcc ctctgccaaa aattatgggg acatcatgaa
     4081 gccccttgag catctgactt ctggctaata aaggaaattt attttcattg caatagtgtg
     4141 ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc atttaaaaca
     4201 tcagaatgag tatttggttt agagtttggc aacatatgcc catatgctgg ctgccatgaa
     4261 caaaggttgg ctataaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt
     4321 attccataga aaagccttga cttgaggtta gatttttttt atattttgtt ttgtgttatt
     4381 tttttcttta acatccctaa aattttcctt acatgtttta ctagccagat ttttcctcct
     4441 ctcctgacta ctcccagtca tagctgtccc tcttctctta tggagatccc tcgacctgca
     4501 ccgtcgacca gctggtcgac ggtgcaccgt cgaccagctt ggcgtaatca tggtcatagc
     4561 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4621 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     4681 cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc tcaattagtc
     4741 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     4801 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     4861 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     4921 aaagctaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     4981 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     5041 gtatcttatc atgtctggat ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5101 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5161 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5221 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5281 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5341 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5401 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5461 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5521 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5581 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5641 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5701 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5761 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5821 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5881 gatctcaa
//
