LOCUS       sequence-152085-        5641 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Csy4 with R115A and R119A mutations from the CBA promoter.
            Cloned into an Adeno-Associated Virus backbone for packaging into 
            AAV..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5641)
  AUTHORS   Borchardt EK, Vandoros LA, Huang M, Lackey PE, Marzluff WF, Asokan A
  TITLE     Controlling mRNA stability and translation with the CRISPR 
            endoribonuclease Csy4.
  JOURNAL   RNA. 2015 Nov;21(11):1921-30. doi: 10.1261/rna.051227.115. Epub 2015
            Sep 9.
  PUBMED    26354771
REFERENCE   2  (bases 1 to 5641)
  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"; doi: 
            "10.1261/rna.051227.115"; journalName: "RNA"; date: "2015-11"; 
            volume: "21"; issue: "11"; pages: "1921-30"
COMMENT     Sequence Label: TR-Csy4-R115A/R119A
FEATURES             Location/Qualifiers
     source          1..5641
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             7..570
                     /codon_start=1
                     /product="CRISPR-associated endoribonuclease responsible 
                     for pre-crRNA processing in Pseudomonas aeruginosa 
                     (Haurwitz et al., 2010)"
                     /label=Csy4
                     /translation="MDHYLDIRLRPDPEFPPAQLMSVLFGKLHQALVAQGGDRIGVSFP
                     DLDESRSRLGERLRIHASADDLRALLARPWLEGLRDHLQFGEPAVVPHPTPYRQVSRVQ
                     AKSNPERLRRALMRAHDLSEEEARKRIPDTVARALDLPFVTLRSQSTGQHFRLFIRHGP
                     LQVTAEEGGFTCYGLSKGGFVPWF"
     polyA_signal    594..715
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(605..624)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     659..678
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      1066..1521
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1153..1172)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1363..1384
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1588..1607)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1707..1729
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1767..1785)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1853..1957
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1958..2026,2027..2818)
                     /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     complement(2176..2195)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2989..3577
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3478..3497
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        4012..4391
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4393..4670
                     /label=chicken beta-actin promoter
     primer_bind     5613..5635
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
ORIGIN
        1 tctagaatgg accactacct cgacattcgc ttgcgaccgg acccggaatt tcccccggcg
       61 caactcatga gcgtgctctt cggcaagctc caccaggccc tggtggcaca gggcggggac
      121 aggatcggcg tgagcttccc cgacctcgac gaaagccgct cccggctggg cgagcgcctg
      181 cgcattcatg cctcggcgga cgaccttcgt gccctgctcg cccggccctg gctggaaggg
      241 ttgcgggacc atctgcaatt cggagaaccg gcagtcgtgc ctcaccccac accgtaccgt
      301 caggtcagtc gggttcaggc gaaaagcaat ccggaacgcc tgcggcgggc cctcatgcgc
      361 gcccacgatc tgagtgagga ggaggctcgg aaacgcattc ccgatacggt cgcgagagcc
      421 ttggacctgc ccttcgtcac gctacgcagc cagagcaccg gacagcactt ccgtctcttc
      481 atccgccacg ggccgttgca ggtgacggca gaggaaggag gattcacctg ttacgggttg
      541 agcaaaggag gtttcgttcc ctggttctga gcggccgctt cgagcagaca tgataagata
      601 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
      661 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
      721 caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag
      781 caagtaaaac ctctacaaat gtggtaaaat cgataaggat ctaggaaccc ctagtgatgg
      841 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg
      901 ggcgtcgggc gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag
      961 tggccaaccc cccccccccc ccccctgcag cctggcgtaa tagcgaagag gcccgcaccg
     1021 atcgcccttc ccaacagttg cgtagcctga atggcgaatg gcgcgacgcg ccctgtagcg
     1081 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     1141 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     1201 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     1261 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     1321 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     1381 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     1441 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     1501 aaatattaac gtttacaatt tcctgatgcg ctattttctc cttacgcatc tgtgcggtat
     1561 ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     1621 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     1681 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     1741 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     1801 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     1861 ccctatttgt ttatttttct aaatactttc aaatatgtat ccgctcatga gacaataacc
     1921 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     1981 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     2041 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     2101 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     2161 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     2221 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     2281 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     2341 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     2401 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     2461 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     2521 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     2581 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     2641 tattgcggat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     2701 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     2761 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     2821 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     2881 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     2941 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3001 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     3061 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     3121 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     3181 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     3241 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     3301 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     3361 gagataccta cagcgtgagc attgagaaag cgccacgctt cccgaaggga gaaaggcgga
     3421 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     3481 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     3541 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     3601 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     3661 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctgggg gggggggggg
     3721 gggttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     3781 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagagaggga
     3841 gtggccaact ccatcactag gggttcctag atcttcaata ttggccatta gccatattat
     3901 tcattggtta tatagcataa atcaatattg gatattggcc attgcatacg ttgtatctat
     3961 atcataatat gtacatttat attggctcat gtccaatatg accgccatgt tggcattgat
     4021 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
     4081 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
     4141 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
     4201 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
     4261 atatgccaag tccgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
     4321 cccagtacat gaccttacgg gactttccta cttggcagta catctacgta ttagtcatcg
     4381 ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct
     4441 ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg
     4501 gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg ggcggggcga
     4561 ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg
     4621 cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg
     4681 cgacgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc
     4741 tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt
     4801 aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag
     4861 gggctccggg agggcccttt gtgcgggggg gagcggctcg gggggtgcgt gcgtgtgtgt
     4921 gtgcgtgggg agcgccgcgt gcggcccgcg ctgcccggcg gctgtgagcg ctgcgggcgc
     4981 ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc
     5041 ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg
     5101 gggtgagcag ggggtatggg cgcggcggtc gggctgtaac ccccccctgc acccccctcc
     5161 ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc gtggcgcggg
     5221 gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg cggggccgcc
     5281 tcgggccggg gagggctcgg gggaggggcg cggcggcccc cggagcgccg gcggctgtcg
     5341 aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg cgcagggact
     5401 tactttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca ccccctctag
     5461 cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg agggccttcg
     5521 tgcgtcgccg cgccgccgtc cccttctccc tctccagcct cggggctgtc cgcgggggga
     5581 cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg
     5641 c
//
