LOCUS       sequence-376095-        5330 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Reprogrammed short tracrRNA generator for hijacking RFP mRNA as 
            CRISPR RNA (target site 1).
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: pLY237
FEATURES             Location/Qualifiers
     source          1..5330
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     9..31
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    56..77
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     CDS             complement(184..1062)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1089..1373
                     /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     1296..1315
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     protein_bind    1444..1462
                     /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    1470..1488
                     /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    1492..1510
                     /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    1587..1607
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      1662..1756
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(1877..1896)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1888..2703
                     /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            2870..2978
                     /note="incP origin of transfer"
     CDS             complement(3039..4187)
                     /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      4580..5111
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(5238..5324)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ttaattaaag cggataacaa tttcacacag gaggccgcct aggccgcggc cgcgcgaatt
       61 cgcggccgct tctagagcct agttgtcttc atgcatgaag acaaaattaa tactagaggg
      121 accaaaacga aaaaagacgc tcgaaagcgt ctcttttctg gaatttggta ccgagctcta
      181 gtattatgac aacttgacgg ctacatcatt cactttttct tcacaaccgg cacggaactc
      241 gctcgggctg gccccggtgc attttttaaa tacccgcgag aaatagagtt gatcgtcaaa
      301 accaacattg cgaccgacgg tggcgatagg catccgggtg gtgctcaaaa gcagcttcgc
      361 ctggctgata cgttggtcct cgcgccagct taagacgcta atccctaact gctggcggaa
      421 aagatgtgac agacgcgacg gcgacaagca aacatgctgt gcgacgctgg cgatatcaaa
      481 attgctgtct gccaggtgat cgctgatgta ctgacaagcc tcgcgtaccc gattatccat
      541 cggtggatgg agcgactcgt taatcgcttc catgcgccgc agtaacaatt gctcaagcag
      601 atttatcgcc agcagctccg aatagcgccc ttccccttgc ccggcgttaa tgatttgccc
      661 aaacaggtcg ctgaaatgcg gctggtgcgc ttcatccggg cgaaagaacc ccgtattggc
      721 aaatattgac ggccagttaa gccattcatg ccagtaggcg cgcggacgaa agtaaaccca
      781 ctggtgatac cattcgcgag cctccggatg acgaccgtag tgatgaatct ctcctggcgg
      841 gaacagcaaa atatcacccg gtcggcaaac aaattctcgt ccctgatttt tcaccacccc
      901 ctgaccgcga atggtgagat tgagaatata acctttcatt cccagcggtc ggtcgataaa
      961 aaaatcgaga taaccgttgg cctcaatcgg cgttaaaccc gccaccagat gggcattaaa
     1021 cgagtatccc ggcagcaggg gatcattttg cgcttcagcc atacttttca tactcccgcc
     1081 attcagagaa gaaaccaatt gtccatattg catcagacat tgccgtcact gcgtctttta
     1141 ctggctcttc tcgctaacca aaccggtaac cccgcttatt aaaagcattc tgtaacaaag
     1201 cgggaccaaa gccatgacaa aaacgcgtaa caaaagtgtc tataatcacg gcagaaaagt
     1261 ccacattgat tatttgcacg gcgtcacact ttgctatgcc atagcatttt tatccataag
     1321 attagcggat cctacctgac gctttttatc gcaactctct actgtttctc catccttgta
     1381 gatgaacaag tgccgttaag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
     1441 tgcgggccct gaagaagggc ccaaggctag ggccctgaag aagggcccgc agggccctga
     1501 agaagggccc ttttttttac tagagctcgg taccaaattc cagaaaagag gcctcccgaa
     1561 aggggggcct tttttcgttt tggtcctact agtagcggcc gctgcaggca tgcaagcttg
     1621 cggccgcgtc gtgactggga aaaccctggc gactagtctt ggactcctgt tgatagatcc
     1681 agtaatgacc tcagaactcc atctggattt gttcagaacg ctcggttgcc gccgggcgtt
     1741 ttttattggt gagaatccag gggtccccaa taattacgat ttaaatttgt gtctcaaaat
     1801 ctctgatgtt acattgcaca agataaaaat atatcatcat gaacaataaa actgtctgct
     1861 tacataaaca gtaatacaag gggtgttatg agccatattc agcgtgaaac gagctgtagc
     1921 cgtccgcgtc tgaacagcaa catggatgcg gatctgtatg gctataaatg ggcgcgtgat
     1981 aacgtgggtc agagcggcgc gaccatttat cgtctgtatg gcaaaccgga tgcgccggaa
     2041 ctgtttctga aacatggcaa aggcagcgtg gcgaacgatg tgaccgatga aatggtgcgt
     2101 ctgaactggc tgaccgaatt tatgccgctg ccgaccatta aacattttat tcgcaccccg
     2161 gatgatgcgt ggctgctgac caccgcgatt ccgggcaaaa ccgcgtttca ggtgctggaa
     2221 gaatatccgg atagcggcga aaacattgtg gatgcgctgg ccgtgtttct gcgtcgtctg
     2281 catagcattc cggtgtgcaa ctgcccgttt aacagcgatc gtgtgtttcg tctggcccag
     2341 gcgcagagcc gtatgaacaa cggcctggtg gatgcgagcg attttgatga tgaacgtaac
     2401 ggctggccgg tggaacaggt gtggaaagaa atgcataaac tgctgccgtt tagcccggat
     2461 agcgtggtga cccacggcga ttttagcctg gataacctga ttttcgatga aggcaaactg
     2521 attggctgca ttgatgtggg ccgtgtgggc attgcggatc gttatcagga tctggccatt
     2581 ctgtggaact gcctgggcga atttagcccg agcctgcaaa aacgtctgtt tcagaaatat
     2641 ggcattgata atccggatat gaacaaactg caatttcatc tgatgctgga tgaatttttc
     2701 taataattaa ttggaccgcg gtccgcgcgt tgtccttttc cgctgcataa ccctgcttcg
     2761 gggtcattat agcgattttt tcggtatatc catccttttt cgcacgatat acaggatttt
     2821 gccaaagggt tcgtgtagac tttccttggt gtatccaacg gcgtcagccg ggcaggatag
     2881 gtgaagtagg cccacccgcg agcgggtgtt ccttcttcac tgtcccttat tcgcacctgg
     2941 cggtgctcaa cgggaatcct gctctgcgag gctggccgta ggccggccgc gatgcaggtg
     3001 gctgctgaac ccccagccgg aactgacccc acaaggccct agcgtttgca atgcaccagg
     3061 tcatcattga cccaggcgtg ttccaccagg ccgctgcctc gcaactcttc gcaggcttcg
     3121 ccgacctgct cgcgccactt cttcacgcgg gtggaatccg atccgcacat gaggcggaag
     3181 gtttccagct tgagcgggta cggctcccgg tgcgagctga aatagtcgaa catccgtcgg
     3241 gccgtcggcg acagcttgcg gtacttctcc catatgaatt tcgtgtagtg gtcgccagca
     3301 aacagcacga cgatttcctc gtcgatcagg acctggcaac gggacgtttt cttgccacgg
     3361 tccaggacgc ggaagcggtg cagcagcgac accgattcca ggtgcccaac gcggtcggac
     3421 gtgaagccca tcgccgtcgc ctgtaggcgc gacaggcatt cctcggcctt cgtgtaatac
     3481 cggccattga tcgaccagcc caggtcctgg caaagctcgt agaacgtgaa ggtgatcggc
     3541 tcgccgatag gggtgcgctt cgcgtactcc aacacctgct gccacaccag ttcgtcatcg
     3601 tcggcccgca gctcgacgcc ggtgtaggtg atcttcacgt ccttgttgac gtggaaaatg
     3661 accttgtttt gcagcgcctc gcgcgggatt ttcttgttgc gcgtggtgaa cagggcagag
     3721 cgggccgtgt cgtttggcat cgctcgcatc gtgtccggcc acggcgcaat atcgaacaag
     3781 gaaagctgca tttccttgat ctgctgcttc gtgtgtttca gcaacgcggc ctgcttggct
     3841 tcgctgacct gttttgccag gtcctcgccg gcggtttttc gcttcttggt cgtcatagtt
     3901 cctcgcgtgt cgatggtcat cgacttcgcc aaacctgccg cctcctgttc gagacgacgc
     3961 gaacgctcca cggcggccga tggcgcgggc agggcagggg gagccagttg cacgctgtcg
     4021 cgctcgatct tggccgtagc ttgctggact atcgagccga cggactggaa ggtttcgcgg
     4081 ggcgcacgca tgacggtgcg gcttgcgatg gtttcggcat cctcggcgga aaaccccgcg
     4141 tcgatcagtt cttgcctgta tgccttccgg tcaaacgtcc gattcattca ccctccttgc
     4201 gggattgccc cggaattaat tccccggatc gatccgtcga tcttgatccc ctgcgccatc
     4261 agatccttgg cggcaagaaa gccatccagt ttactttgca gggcttccca accttaccag
     4321 agggcgcccc agctggcaat tccggttcgc ttgctgtcca taaaaccgcc cagtctagct
     4381 atcgccatgt aagcccactg caagctacct gctttctctt tgcgcttgcg ttttcccttg
     4441 tccagatagc ccagtagctg acattcatcc ggggtcagca ccgtttctgc ggactggctt
     4501 tctacgtggc tgccattttt ggggtgaggc cgttcgcggc cgaggggcgc agcccctggg
     4561 gggatgggag gcccgcgtta gcgggccggg agggttcgag aagggggggc accccccttc
     4621 ggcgtgcgcg gtcacgcgca cagggcgcag ccctggttaa aaacaaggtt tataaatatt
     4681 ggtttaaaag caggttaaaa gacaggttag cggtggccga aaaacgggcg gaaacccttg
     4741 caaatgctgg attttctgcc tgtggacagc ccctcaaatg tcaataggtg cgcccctcat
     4801 ctgtcagcac tctgcccctc aagtgtcaag gatcgcgccc ctcatctgtc agtagtcgcg
     4861 cccctcaagt gtcaataccg cagggcactt atccccaggc ttgtccacat catctgtggg
     4921 aaactcgcgt aaaatcaggc gttttcgccg atttgcgagg ctggccagct ccacgtcgcc
     4981 ggccgaaatc gagcctgccc ctcatctgtc aacgccgcgc cgggtgagtc ggcccctcaa
     5041 gtgtcaacgt ccgcccctca tctgtcagtg agggccaagt tttccgcgag gtatccacaa
     5101 cgccggcggc cctacatggc tctgctgtag tgagtgggtt gcgctccggc agcggtcctg
     5161 atcccccgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggcgcgccca
     5221 gctgtctagg gcggcggatt tgtcctactc aggagagcgt tcaccgacaa acaacagata
     5281 aaacgaaagg cccagtcttt cgactgagcc tttcgtttta tttgatgcct
//
