LOCUS       sequence-308571-        7824 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses type I-F P. aeruginosa cascade (PaeCascade) proteins 
            Csy3-VPR and Csy4 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7824)
  AUTHORS   Chen Y, Liu J, Zhi S, Zheng Q, Ma W, Huang J, Liu Y, Liu D, Liang P,
            Songyang Z
  TITLE     Repurposing type I-F CRISPR-Cas system as a transcriptional 
            activation tool in human cells.
  JOURNAL   Nat Commun. 2020 Jun 19;11(1):3136. doi: 10.1038/s41467-020-16880-8.
  PUBMED    32561716
REFERENCE   2  (bases 1 to 7824)
  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: "2020-06-19"; volume: "11(1):3136. doi"; pages: " 
            10.1038/s41467-020-16880-8"
COMMENT     Sequence Label: pCsy3-VPR-Csy4
FEATURES             Location/Qualifiers
     source          1..7824
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        154..533
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        534..737
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     688..708
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             767..787
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1835..1855
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1880..2029
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             2468..2824
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             3419..3499
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(3527..3544)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3533..3740
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3747..4246
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3782..3805)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4179..4198
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4275..4295
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4863..4928
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    4963..5084
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4974..4993)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5028..5047
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   5087..5227
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5087..5216
                     /label=AAV2 ITR
     rep_origin      5302..5757
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5389..5408)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5599..5620
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5774..5793)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5893..5915
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5953..5971)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6039..6143
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6144..6212,6213..7004)
                     /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(6362..6381)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7175..7763
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7664..7683
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat
      181 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      241 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      301 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      361 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      421 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      481 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      541 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      601 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      661 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      721 tctatataag cagagctctc tggctaacta ccggcgcgcc accatgccga agaagaagag
      781 gaaagtttcc aatgcatcca agccaatact gagcaccgct tccgtcctcg ccttcgaacg
      841 taagctcgac ccttccgacg cccttatgag cgctggcgcc tgggcgcaac gcgacgcctc
      901 gcaggaatgg ccggccgtga ccgtgcgcga gaagtccgta cgcggcacca tctccaaccg
      961 cctcaagacc aaggaccgtg acccggccaa gctggacgcc tcgatccagt cgcccaacct
     1021 gcagacggtg gacgtggcca acctgccgag cgacgccgac accctcaagg tccgcttcac
     1081 cctccgtgtg ctcggcggcg ccggaacacc gtctgcctgc aacgacgcgg cctaccgtga
     1141 caagctgctg caaacggtcg ccacctacgt gaacgatcag ggcttcgccg agctggctcg
     1201 tcgttatgcg cacaacctgg ccaacgcccg cttcctgtgg cgcaaccggg tgggcgcgga
     1261 agcggtggaa gtccgtatca accatatccg ccagggcgag gtggctcgcg cctggcgctt
     1321 cgacgccctg gccatcggct tgcgcgactt caaggccgac gccgaactgg acgcgctcgc
     1381 cgaactgatc gccagcggtc tctcaggcag tgggcatgtc ctgctcgaag tggtcgcctt
     1441 cgcccgtatc ggcgacggcc aggaagtctt cccctcccag gaactgatcc tcgacaaagg
     1501 cgacaagaaa ggccagaaga gcaagaccct gtacagcgta cgcgatgccg cggccatcca
     1561 ctcgcagaag atcggcaatg ccctgcgcac catcgatacg tggtatcccg acgaagatgg
     1621 cctcggcccc atcgccgtgg agccctacgg ctccgtcaca tcccagggca aagcctatcg
     1681 ccagcccaag cagaagctgg acttctatac gctgctcgac aactgggtac tgcgcgacga
     1741 ggcgcccgcc gtggagcaac agcattatgt gatcgccaac ctgatccgtg gcggcgtgtt
     1801 cggtgaagcc gaagagaaga cgcgtagagc tgaccccaag aagaagagga aggtggaggc
     1861 cagcggttcc ggacgggctg acgcattgga cgattttgat ctggatatgc tgggaagtga
     1921 cgccctcgat gattttgacc ttgacatgct tggttcggat gcccttgatg actttgacct
     1981 cgacatgctc ggcagtgacg cccttgatga tttcgacctg gacatgctga ttaactctag
     2041 aagccaatac ctcccagaca cagacgacag gcataggatt gaggaaaaaa gaaagaggac
     2101 gtatgaaact ttcaagagca taatgaaaaa aagtccgttc tctggtccta cagaccccag
     2161 gcccccacct agacgaatcg cggttccgag ccgatcatca gcgagtgttc ctaagccagc
     2221 tccacagccc tatccattta cgtctagtct gtctacgatc aactacgacg agttccccac
     2281 tatggtattc ccgtcaggcc agataagcca agctagtgcg ttggcacccg cacccccaca
     2341 ggtgctccca caagctcccg ctcctgcgcc agcaccagct atggttagtg ccctcgcgca
     2401 agcgcctgcc ccggttcctg ttctcgcacc tgggccaccg caggcagttg caccgccagc
     2461 gccaaagccc acacaggcag gggaaggtac gttgagcgaa gcccttctgc aactccaatt
     2521 tgacgatgag gacctcggag cacttcttgg caattcaacg gaccccgcag tatttactga
     2581 tcttgccagc gtggacaatt ccgaatttca acagctcctg aaccagggaa tcccggtggc
     2641 tccccatacg acagagccca tgctcatgga gtacccggag gccattacac gccttgtgac
     2701 gggcgcacag cgaccccctg acccggcacc ggctcccttg ggggctcctg ggcttcccaa
     2761 cgggttgctg tcaggcgacg aggactttag ctcaatcgcc gacatggatt ttagcgccct
     2821 gttgggtagc ggaagtggta gccgcgacag ccgggaaggc atgttccttc cgaaaccgga
     2881 agctggtagc gccataagcg acgtatttga gggtagggag gtgtgtcagc ctaagcggat
     2941 tcgaccattc cacccccctg gctctccttg ggcaaatcga cccctcccgg cgagccttgc
     3001 ccctacgccg accggacccg tgcatgagcc cgtcggtagc ctcacgccag cccccgtgcc
     3061 gcaacctctc gaccctgctc cggcggttac gccagaagca agccaccttt tggaggaccc
     3121 ggacgaagaa acaagccagg cggtaaaggc gctccgcgaa atggccgaca ccgtgatacc
     3181 ccagaaggag gaagccgcca tttgcggcca aatggacctt tcccaccctc caccacgcgg
     3241 ccacctggac gagctgacga ccaccctgga aagcatgacc gaggatttga atctggacag
     3301 cccgttgacc cccgaattga acgaaatcct ggacacgttc ctgaatgatg agtgtttgtt
     3361 gcacgctatg cacatcagca ccgggtttag tatatttgac acaagtttgt ttggatccta
     3421 cccatacgat gttccagatt acgcttaccc atacgatgtt ccagattacg cttacccata
     3481 cgatgttcca gattacgctt aagaattcct agagctcgct gatcagcctc gactgtgcct
     3541 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     3601 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     3661 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagag
     3721 aatagcaggc atgctgggga ggtaccgggt aggggaggcg cttttcccaa ggcagtctgg
     3781 agcatgcgct ttagcagccc cgctgggcac ttggcgctac acaagtggcc tctggcctcg
     3841 cacacattcc acatccaccg gtaggcgcca accggctccg ttctttggtg gccccttcgc
     3901 gccaccttct actcctcccc tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg
     3961 caggacgtga caaatggaag tagcacgtct cactagtctc gtgcagatgg acagcaccgc
     4021 tgagcaatgg aagcgggtag gcctttgggg cagcggccaa tagcagcttt gctccttcgc
     4081 tttctgggct cagaggctgg gaaggggtgg gtccgggggc gggctcaggg gcgggctcag
     4141 gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca ttctgcacgc ttcaaaagcg
     4201 cacgtctgcc gcgctgttct cctcttcctc atctccgggc ctttcgacct gcatcccgcg
     4261 gccgcgccac catgccaaag aagaagcgga aagtcgacca ctatctggac atcagactga
     4321 ggcccgatcc tgagttccct cccgcccagc tgatgagcgt gctgtttggc aagctgcatc
     4381 aggctctggt cgcccaaggc ggagacagaa tcggcgtgtc cttccccgac ctggacgagt
     4441 cccggagtcg cctgggcgag cggctgagaa tccacgccag cgcagacgat ctgcgcgccc
     4501 tgctggcccg gccttggctg gagggcctgc gggatcatct gcagtttggc gagcccgccg
     4561 tggtgccaca cccaacaccc taccgccagg tgagccgcgt gcaggccaag tcaaatcccg
     4621 agagactgcg gcggaggctg atgaggcgac atgatctgag cgaggaggag gccagaaaga
     4681 gaatccccga cacagtggcc agagccctgg atctgccatt tgtgaccctg cggagccaga
     4741 gcactggcca gcatttcaga ctgttcatca gacacgggcc cctgcaggtg acagccgagg
     4801 agggcggatt tacatgctat ggcctgtcta aaggcggctt cgtgccctgg ttccaagctt
     4861 gcgactataa ggaccacgac ggagactaca aggatcatga tattgattac aaagacgatg
     4921 acgataagta agggcccgtg taggctagtc gagcagacat gataagatac attgatgagt
     4981 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     5041 ctattgcttt atttgtaacc attataagct gcaataaaca agttgcagga acccctagtg
     5101 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     5161 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc
     5221 ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     5281 atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     5341 tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt
     5401 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     5461 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     5521 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     5581 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct
     5641 cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg
     5701 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
     5761 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     5821 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     5881 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     5941 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     6001 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     6061 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     6121 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     6181 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     6241 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     6301 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     6361 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     6421 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     6481 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     6541 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     6601 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     6661 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     6721 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     6781 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     6841 ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca gatggtaagc
     6901 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     6961 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     7021 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     7081 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     7141 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     7201 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     7261 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     7321 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     7381 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     7441 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     7501 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     7561 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     7621 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     7681 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     7741 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     7801 tttgctggcc ttttgctcac atgt
//
