LOCUS       sequence-376092-        5330 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Reprogrammed short tracrRNA generator for hijacking RFP mRNA as 
            CRISPR RNA (target site 3).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5330)
  AUTHORS   Liu Y, Pinto F, Wan X, Yang Z, Peng S, Li M, Cooper JM, Xie Z, 
            French CE, Wang B
  TITLE     Reprogrammed tracrRNAs enable repurposing of RNAs as crRNAs and 
            sequence-specific RNA biosensors.
  JOURNAL   Nat Commun. 2022 Apr 11;13(1):1937. doi: 10.1038/s41467-022-29604-x.
  PUBMED    35410423
REFERENCE   2  (bases 1 to 5330)
  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: "Nat 
            Commun."; date: "2022-04-11"; volume: "13(1):1937. doi"; pages: " 
            10.1038/s41467-022-29604-x"
COMMENT     Sequence Label: pLY239
FEATURES             Location/Qualifiers
     source          1..5330
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     10..32
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    57..78
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     CDS             complement(185..1063)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1090..1374
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     1297..1316
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     protein_bind    1445..1463
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    1471..1489
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    1493..1511
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     misc_feature    1588..1608
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      1663..1757
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(1878..1897)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1889..2704
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     oriT            2871..2979
                     /note="incP origin of transfer"
     CDS             complement(3040..4188)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     IVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      4581..5112
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(5239..5325)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 tttaattaaa gcggataaca atttcacaca ggaggccgcc taggccgcgg ccgcgcgaat
       61 tcgcggccgc ttctagagcc tagttgtctt catgcatgaa gacaaaatta atactagagg
      121 gaccaaaacg aaaaaagacg ctcgaaagcg tctcttttct ggaatttggt accgagctct
      181 agtattatga caacttgacg gctacatcat tcactttttc ttcacaaccg gcacggaact
      241 cgctcgggct ggccccggtg cattttttaa atacccgcga gaaatagagt tgatcgtcaa
      301 aaccaacatt gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg
      361 cctggctgat acgttggtcc tcgcgccagc ttaagacgct aatccctaac tgctggcgga
      421 aaagatgtga cagacgcgac ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa
      481 aattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc cgattatcca
      541 tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca
      601 gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcgtta atgatttgcc
      661 caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg
      721 caaatattga cggccagtta agccattcat gccagtaggc gcgcggacga aagtaaaccc
      781 actggtgata ccattcgcga gcctccggat gacgaccgta gtgatgaatc tctcctggcg
      841 ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccctgattt ttcaccaccc
      901 cctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt cggtcgataa
      961 aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa
     1021 acgagtatcc cggcagcagg ggatcatttt gcgcttcagc catacttttc atactcccgc
     1081 cattcagaga agaaaccaat tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt
     1141 actggctctt ctcgctaacc aaaccggtaa ccccgcttat taaaagcatt ctgtaacaaa
     1201 gcgggaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag
     1261 tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt ttatccataa
     1321 gattagcgga tcctacctga cgctttttat cgcaactctc tactgtttct ccataggtgg
     1381 tcttgaccaa gtagcgttaa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
     1441 gtgcgggccc tgaagaaggg cccaaggcta gggccctgaa gaagggcccg cagggccctg
     1501 aagaagggcc ctttttttta ctagagctcg gtaccaaatt ccagaaaaga ggcctcccga
     1561 aaggggggcc ttttttcgtt ttggtcctac tagtagcggc cgctgcaggc atgcaagctt
     1621 gcggccgcgt cgtgactggg aaaaccctgg cgactagtct tggactcctg ttgatagatc
     1681 cagtaatgac ctcagaactc catctggatt tgttcagaac gctcggttgc cgccgggcgt
     1741 tttttattgg tgagaatcca ggggtcccca ataattacga tttaaatttg tgtctcaaaa
     1801 tctctgatgt tacattgcac aagataaaaa tatatcatca tgaacaataa aactgtctgc
     1861 ttacataaac agtaatacaa ggggtgttat gagccatatt cagcgtgaaa cgagctgtag
     1921 ccgtccgcgt ctgaacagca acatggatgc ggatctgtat ggctataaat gggcgcgtga
     1981 taacgtgggt cagagcggcg cgaccattta tcgtctgtat ggcaaaccgg atgcgccgga
     2041 actgtttctg aaacatggca aaggcagcgt ggcgaacgat gtgaccgatg aaatggtgcg
     2101 tctgaactgg ctgaccgaat ttatgccgct gccgaccatt aaacatttta ttcgcacccc
     2161 ggatgatgcg tggctgctga ccaccgcgat tccgggcaaa accgcgtttc aggtgctgga
     2221 agaatatccg gatagcggcg aaaacattgt ggatgcgctg gccgtgtttc tgcgtcgtct
     2281 gcatagcatt ccggtgtgca actgcccgtt taacagcgat cgtgtgtttc gtctggccca
     2341 ggcgcagagc cgtatgaaca acggcctggt ggatgcgagc gattttgatg atgaacgtaa
     2401 cggctggccg gtggaacagg tgtggaaaga aatgcataaa ctgctgccgt ttagcccgga
     2461 tagcgtggtg acccacggcg attttagcct ggataacctg attttcgatg aaggcaaact
     2521 gattggctgc attgatgtgg gccgtgtggg cattgcggat cgttatcagg atctggccat
     2581 tctgtggaac tgcctgggcg aatttagccc gagcctgcaa aaacgtctgt ttcagaaata
     2641 tggcattgat aatccggata tgaacaaact gcaatttcat ctgatgctgg atgaattttt
     2701 ctaataatta attggaccgc ggtccgcgcg ttgtcctttt ccgctgcata accctgcttc
     2761 ggggtcatta tagcgatttt ttcggtatat ccatcctttt tcgcacgata tacaggattt
     2821 tgccaaaggg ttcgtgtaga ctttccttgg tgtatccaac ggcgtcagcc gggcaggata
     2881 ggtgaagtag gcccacccgc gagcgggtgt tccttcttca ctgtccctta ttcgcacctg
     2941 gcggtgctca acgggaatcc tgctctgcga ggctggccgt aggccggccg cgatgcaggt
     3001 ggctgctgaa cccccagccg gaactgaccc cacaaggccc tagcgtttgc aatgcaccag
     3061 gtcatcattg acccaggcgt gttccaccag gccgctgcct cgcaactctt cgcaggcttc
     3121 gccgacctgc tcgcgccact tcttcacgcg ggtggaatcc gatccgcaca tgaggcggaa
     3181 ggtttccagc ttgagcgggt acggctcccg gtgcgagctg aaatagtcga acatccgtcg
     3241 ggccgtcggc gacagcttgc ggtacttctc ccatatgaat ttcgtgtagt ggtcgccagc
     3301 aaacagcacg acgatttcct cgtcgatcag gacctggcaa cgggacgttt tcttgccacg
     3361 gtccaggacg cggaagcggt gcagcagcga caccgattcc aggtgcccaa cgcggtcgga
     3421 cgtgaagccc atcgccgtcg cctgtaggcg cgacaggcat tcctcggcct tcgtgtaata
     3481 ccggccattg atcgaccagc ccaggtcctg gcaaagctcg tagaacgtga aggtgatcgg
     3541 ctcgccgata ggggtgcgct tcgcgtactc caacacctgc tgccacacca gttcgtcatc
     3601 gtcggcccgc agctcgacgc cggtgtaggt gatcttcacg tccttgttga cgtggaaaat
     3661 gaccttgttt tgcagcgcct cgcgcgggat tttcttgttg cgcgtggtga acagggcaga
     3721 gcgggccgtg tcgtttggca tcgctcgcat cgtgtccggc cacggcgcaa tatcgaacaa
     3781 ggaaagctgc atttccttga tctgctgctt cgtgtgtttc agcaacgcgg cctgcttggc
     3841 ttcgctgacc tgttttgcca ggtcctcgcc ggcggttttt cgcttcttgg tcgtcatagt
     3901 tcctcgcgtg tcgatggtca tcgacttcgc caaacctgcc gcctcctgtt cgagacgacg
     3961 cgaacgctcc acggcggccg atggcgcggg cagggcaggg ggagccagtt gcacgctgtc
     4021 gcgctcgatc ttggccgtag cttgctggac tatcgagccg acggactgga aggtttcgcg
     4081 gggcgcacgc atgacggtgc ggcttgcgat ggtttcggca tcctcggcgg aaaaccccgc
     4141 gtcgatcagt tcttgcctgt atgccttccg gtcaaacgtc cgattcattc accctccttg
     4201 cgggattgcc ccggaattaa ttccccggat cgatccgtcg atcttgatcc cctgcgccat
     4261 cagatccttg gcggcaagaa agccatccag tttactttgc agggcttccc aaccttacca
     4321 gagggcgccc cagctggcaa ttccggttcg cttgctgtcc ataaaaccgc ccagtctagc
     4381 tatcgccatg taagcccact gcaagctacc tgctttctct ttgcgcttgc gttttccctt
     4441 gtccagatag cccagtagct gacattcatc cggggtcagc accgtttctg cggactggct
     4501 ttctacgtgg ctgccatttt tggggtgagg ccgttcgcgg ccgaggggcg cagcccctgg
     4561 ggggatggga ggcccgcgtt agcgggccgg gagggttcga gaaggggggg cacccccctt
     4621 cggcgtgcgc ggtcacgcgc acagggcgca gccctggtta aaaacaaggt ttataaatat
     4681 tggtttaaaa gcaggttaaa agacaggtta gcggtggccg aaaaacgggc ggaaaccctt
     4741 gcaaatgctg gattttctgc ctgtggacag cccctcaaat gtcaataggt gcgcccctca
     4801 tctgtcagca ctctgcccct caagtgtcaa ggatcgcgcc cctcatctgt cagtagtcgc
     4861 gcccctcaag tgtcaatacc gcagggcact tatccccagg cttgtccaca tcatctgtgg
     4921 gaaactcgcg taaaatcagg cgttttcgcc gatttgcgag gctggccagc tccacgtcgc
     4981 cggccgaaat cgagcctgcc cctcatctgt caacgccgcg ccgggtgagt cggcccctca
     5041 agtgtcaacg tccgcccctc atctgtcagt gagggccaag ttttccgcga ggtatccaca
     5101 acgccggcgg ccctacatgg ctctgctgta gtgagtgggt tgcgctccgg cagcggtcct
     5161 gatcccccgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggcgcgccc
     5221 agctgtctag ggcggcggat ttgtcctact caggagagcg ttcaccgaca aacaacagat
     5281 aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt atttgatgcc
//
