LOCUS       sequence-364194-        2517 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constituitvely expressed CasRx crRNA cloning vector, extended 3' 
            terminator region.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2517)
  AUTHORS   Otoupal PB, Cress BF, Doudna JA, Schoeniger JS
  TITLE     CRISPR-RNAa: targeted activation of translation using dCas13 fusions
            to translation initiation factors.
  JOURNAL   Nucleic Acids Res. 2022 Aug 11. pii: 6660959. doi: 
            10.1093/nar/gkac680.
  PUBMED    35950485
REFERENCE   2  (bases 1 to 2517)
  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: "Nucleic 
            Acids Res. 2022 Aug 11. pii: 6660959. doi: 10.1093/nar/gkac680."
COMMENT     Sequence Label: pPBO.ACT004
FEATURES             Location/Qualifiers
     source          1..2517
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        25..59
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        60..89
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     CDS             118..147
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             163..180
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(236..253)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      406..452
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(505..522)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(676..1264)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(756..775)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1435..2226,2227..2295))
                     /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     2058..2077
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2296..2400)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2468..2486
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(join(2490..2517,1..16))
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
ORIGIN
        1 cgctaaggat gatttctgga attcttgaca gctagctcag tcctaggtat aatactagta
       61 acccctacca actggtcggg gtttgaaacg gagacgaacg tctcaaagct tgggcccgaa
      121 caaaaactca tctcagaaga ggatctgaat agcgccgtcg accatcatca tcatcatcat
      181 tgagtttaaa cggtctccag cttggctgtt ttggcggatg agagaagatt ttcagcctga
      241 tacagattaa atcagaacgc agaagcggtc tgataaaaca gaatttgcct ggcggcagta
      301 gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt agcgccgatg
      361 gtagtgtggg gtctccccat gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag
      421 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa ctggatcctt
      481 actcgagtct agactgcagg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
      541 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
      601 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
      661 cgcgttgctg gcgtttttcc acaggctccg cccccctgac gagcatcaca aaaatcgacg
      721 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
      781 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
      841 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
      901 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
      961 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     1021 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     1081 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     1141 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     1201 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     1261 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     1321 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     1381 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     1441 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     1501 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     1561 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     1621 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     1681 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     1741 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     1801 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     1861 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     1921 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     1981 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     2041 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     2101 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     2161 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     2221 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     2281 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     2341 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     2401 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     2461 ctataaaaat aggcgtatca cgaggcagaa tttcagataa aaaaaatcct tagcttt
//
