LOCUS       sequence-451479-        6240 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 membrane RFP reporter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6240)
  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 6240)
  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-3xControlgRNA
FEATURES             Location/Qualifiers
     source          1..6240
                     /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..3764
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     ncRNA           3777..3813
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        3814..3843
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     ncRNA           3867..3934
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     ncRNA           3957..3993
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        3994..4023
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     ncRNA           4047..4114
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     ncRNA           4137..4173
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        4174..4203
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     ncRNA           4227..4294
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(4394..4413)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4459..4514
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4513..4532)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4905..4921)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4905..4921)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4918..4940)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4929..4945
                     /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(4953..4959,4960..4977,4978..4983))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4998..5019
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5078..5273
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5124..5259
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5279..5413
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5316..5335)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5370..5389
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5481..5498)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5652..6240)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5732..5751)
                     /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 ctccggcggc tctggcggca tggcaagcag cgaagatgtt attaaagaat
     3121 ttatgcggtt taaagtccga atggaaggat cagtaaatgg acatgaattt gaaattgaag
     3181 gagaaggtga aggtcgacca tatgaaggaa cgcaaactgc taaactcaaa gtcacgaaag
     3241 gtggtccatt gccgtttgct tgggatatac tctctcccca atttcaatat ggatcaaaag
     3301 catatgttaa acatcctgca gatattcctg attatctgaa actcagcttt ccagaaggct
     3361 ttaaatggga acgggtaatg aattttgaag atggtggggt tgttacagtc acccaagatt
     3421 ctagtcttca agatggggaa tttatatata aagtcaaatt gcggggcaca aattttccaa
     3481 gcgatgggcc agtcatgcaa aagaaaacaa tgggatggga agcatctaca gaaaggatgt
     3541 atccagaaga tggagctctc aaaggggaaa ttaaaatgag attgaaactc aaagatggcg
     3601 ggcattatga tgcagaagtg aaaacgactt atatggcgaa gaaacccgtt cagctgcctg
     3661 gggcttataa aacagatatt aaactcgata ttactagcca taatgaagat tacacaatag
     3721 tcgagcaata tgaacgggca gaagggcggc attcaacagg agctaccggt cagtgcctga
     3781 tgagtccgtg aggacgaaac gagtaagctc gtcaacccct accaactggt cggggtttga
     3841 aacctcttac ctcagttaca atttatggcc ggcatggtcc cagcctcctc gctggcgccg
     3901 gctgggcaac atgcttcggc atggcgaatg ggacatcgat acagcgcaaa attaggctga
     3961 tgagtccgtg aggacgaaac gagtaagctc gtcaacccct accaactggt cggggtttga
     4021 aacctcttac ctcagttaca atttatggcc ggcatggtcc cagcctcctc gctggcgccg
     4081 gctgggcaac atgcttcggc atggcgaatg ggacaagctt acagcgcaaa attaggctga
     4141 tgagtccgtg aggacgaaac gagtaagctc gtcaacccct accaactggt cggggtttga
     4201 aacctcttac ctcagttaca atttatggcc ggcatggtcc cagcctcctc gctggcgccg
     4261 gctgggcaac atgcttcggc atggcgaatg ggactctaga tgagcggccg caattcactc
     4321 ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca
     4381 aataccactg agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg
     4441 agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt
     4501 ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg
     4561 agtatttggt ttagagtttg gcaacatatg cccatatgct ggctgccatg aacaaaggtt
     4621 ggctataaag aggtcatcag tatatgaaac agccccctgc tgtccattcc ttattccata
     4681 gaaaagcctt gacttgaggt tagatttttt ttatattttg ttttgtgtta tttttttctt
     4741 taacatccct aaaattttcc ttacatgttt tactagccag atttttcctc ctctcctgac
     4801 tactcccagt catagctgtc cctcttctct tatggagatc cctcgacctg caccgtcgac
     4861 cagctggtcg acggtgcacc gtcgaccagc ttggcgtaat catggtcata gctgtttcct
     4921 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4981 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     5041 gctttccagt cgggaaacct gtcgtgccag cggatccgca tctcaattag tcagcaacca
     5101 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     5161 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     5221 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctaa
     5281 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     5341 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     5401 tcatgtctgg atccgctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5461 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5521 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5581 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5641 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5701 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5761 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5821 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     5881 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5941 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     6001 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     6061 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     6121 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     6181 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
//
