LOCUS       sequence-361297-        3120 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constituitvely expressed CasRx crRNA cloning vector, truncated 3' 
            terminator region, with eGFP for cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3120)
  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 3120)
  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.ACT006
FEATURES             Location/Qualifiers
     source          1..3120
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..55
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        56..85
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     RBS             262..273
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             280..993
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      1005..1051
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1104..1121)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1275..1863)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1355..1374)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2034..2825,2826..2894))
                     /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     2657..2676
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2895..2999)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3067..3085
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(join(3089..3120,1..12))
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
ORIGIN
        1 aaggatgatt tctggaattc ttgacagcta gctcagtcct aggtataata ctagtaaccc
       61 ctaccaactg gtcggggttt gaaacggaga cgaccggcat tggtcttcag accgaaagtg
      121 aaacgtgatt tcatgcgtca ttttgaacat tttgtaaatc ttatttaata atgtgtgcgg
      181 caattcacat ttaatttatg aatgttttct taacatcgcg gcaactcaag aaacggcagg
      241 ttcggatctt agctactaga gaaagaggag aaatactaga tgcgtaaagg cgaagagctg
      301 ttcactggtg tcgtccctat tctggtggaa ctggatggtg atgtcaacgg tcataagttt
      361 tccgtgcgtg gcgagggtga aggtgacgca actaatggta aactgacgct gaagttcatc
      421 tgtactactg gtaaactgcc ggttccttgg ccgactctgg taacgacgct gacttatggt
      481 gttcagtgct ttgctcgtta tccggaccat atgaagcagc atgacttctt caagtccgcc
      541 atgccggaag gctatgtgca ggaacgcacg atttccttta aggatgacgg cacgtacaaa
      601 acgcgtgcgg aagtgaaatt tgaaggcgat accctggtaa accgcattga gctgaaaggc
      661 attgacttta aagaggacgg caatatcctg ggccataagc tggaatacaa ttttaacagc
      721 cacaatgttt acatcaccgc cgataaacaa aaaaatggca ttaaagcgaa ttttaaaatt
      781 cgccacaacg tggaggatgg cagcgtgcag ctggctgatc actaccagca aaacactcca
      841 atcggtgatg gtcctgttct gctgccagac aatcactatc tgagcacgca aagcgttctg
      901 tctaaagatc cgaacgagaa acgcgatcat atggttctgc tggagttcgt aaccgcagcg
      961 ggcatcacgc atggtatgga tgaactgtac aaatgaacgt ctcacaaata aaacgaaagg
     1021 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac tggatcctta
     1081 ctcgagtcta gactgcaggc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     1141 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     1201 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     1261 gcgttgctgg cgtttttcca caggctccgc ccccctgacg agcatcacaa aaatcgacgc
     1321 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     1381 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     1441 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     1501 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     1561 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     1621 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     1681 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     1741 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     1801 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     1861 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     1921 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     1981 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     2041 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     2101 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     2161 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     2221 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     2281 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     2341 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     2401 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     2461 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     2521 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     2581 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     2641 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     2701 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     2761 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     2821 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     2881 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     2941 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     3001 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     3061 tataaaaata ggcgtatcac gaggcagaat ttcagataaa aaaaatcctt agctttcgct
//
