LOCUS       sequence-451495-        5720 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ubiquitous expression plasmid with CAG promoter (CMV immediate early
            enhancer, chicken beta actin promoter), three Control 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 5720)
  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 5720)
  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-3xControlgRNA
FEATURES             Location/Qualifiers
     source          1..5720
                     /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"
     regulatory      2957..2966
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3089..3136
                     /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             3143..3190
                     /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             3197..3244
                     /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           3257..3293
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        3294..3323
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     ncRNA           3347..3414
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     ncRNA           3437..3473
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        3474..3503
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     ncRNA           3527..3594
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     ncRNA           3617..3653
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        3654..3683
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     ncRNA           3707..3774
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(3874..3893)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3939..3994
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3993..4012)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4385..4401)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4385..4401)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4398..4420)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4409..4425
                     /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(4433..4439,4440..4457,4458..4463))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4478..4499
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4558..4753
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4604..4739
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4759..4893
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4796..4815)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4850..4869
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4961..4978)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5132..5720)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5212..5231)
                     /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 ccatgggatg cattaaatct aagaggaaag ataacttgaa
     3001 cgatgacgaa gctgcaatgg ggtgtattaa atccaaaagg aaagataatt tgaatgatga
     3061 tgaagcgggt agtggtggaa gtggtggtag ggatcatatg gtgctccacg aatacgtgaa
     3121 cgccgccgga ataactggtg ggcgcgatca tatggtgtta cacgaatacg tgaacgcagc
     3181 cggcatcacc ggcggacgcg atcatatggt tttgcacgaa tacgtgaacg ccgcgggaat
     3241 cactaccggt cagtgcctga tgagtccgtg aggacgaaac gagtaagctc gtcaacccct
     3301 accaactggt cggggtttga aacctcttac ctcagttaca atttatggcc ggcatggtcc
     3361 cagcctcctc gctggcgccg gctgggcaac atgcttcggc atggcgaatg ggacatcgat
     3421 acagcgcaaa attaggctga tgagtccgtg aggacgaaac gagtaagctc gtcaacccct
     3481 accaactggt cggggtttga aacctcttac ctcagttaca atttatggcc ggcatggtcc
     3541 cagcctcctc gctggcgccg gctgggcaac atgcttcggc atggcgaatg ggacaagctt
     3601 acagcgcaaa attaggctga tgagtccgtg aggacgaaac gagtaagctc gtcaacccct
     3661 accaactggt cggggtttga aacctcttac ctcagttaca atttatggcc ggcatggtcc
     3721 cagcctcctc gctggcgccg gctgggcaac atgcttcggc atggcgaatg ggactctaga
     3781 tgagcggccg caattcactc ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt
     3841 ggccaatgcc ctggctcaca aataccactg agatcttttt ccctctgcca aaaattatgg
     3901 ggacatcatg aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat
     3961 tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa
     4021 tcatttaaaa catcagaatg agtatttggt ttagagtttg gcaacatatg cccatatgct
     4081 ggctgccatg aacaaaggtt ggctataaag aggtcatcag tatatgaaac agccccctgc
     4141 tgtccattcc ttattccata gaaaagcctt gacttgaggt tagatttttt ttatattttg
     4201 ttttgtgtta tttttttctt taacatccct aaaattttcc ttacatgttt tactagccag
     4261 atttttcctc ctctcctgac tactcccagt catagctgtc cctcttctct tatggagatc
     4321 cctcgacctg caccgtcgac cagctggtcg acggtgcacc gtcgaccagc ttggcgtaat
     4381 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4441 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     4501 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag cggatccgca
     4561 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     4621 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     4681 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     4741 aggcttttgc aaaaagctaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     4801 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     4861 aaactcatca atgtatctta tcatgtctgg atccgctgca ttaatgaatc ggccaacgcg
     4921 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     4981 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5041 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5101 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5161 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5221 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5281 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5341 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5401 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5461 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5521 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5581 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5641 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5701 gcagaaaaaa aggatctcaa
//
