LOCUS       sequence-308572-        7350 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For cloning and expression of Type I-F P. aeruginosa (PaeCascade) 
            crRNA in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7350)
  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 7350)
  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: pCsy-crRNA-EV
FEATURES             Location/Qualifiers
     source          1..7350
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..227
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             228..408
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    455..580
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1073..1306
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1491..1535
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        1713..1961
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1713..1733
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1884..1903
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    1963..1982
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    2023..2042
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     misc_feature    2101..2218
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2287..2498
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2531..2587
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2588..3187
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2588..2607)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3084..3104
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3203..3791
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3256..3276)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3674..3685)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3863..4096
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4174..4295
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4211..4230)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4265..4284
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4335..4470
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4490..4509)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4491..4509)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4519..4536)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4519..4535)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4528..4550)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4677..5132
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4764..4783)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4974..4995
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5158..5262
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5263..5331,5332..6123)
                     /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(5481..5500)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6294..6882
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6783..6802
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7036..7053
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7170..7191
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7206..7211,7212..7229,7230..7236)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7244..7260
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     7249..7271
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7268..7284
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7268..7284
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7303..7323
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7305..7323
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg
       61 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc
      121 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc
      181 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt
      241 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
      301 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
      361 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
      421 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc
      481 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
      541 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
      601 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
      661 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
      721 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
      781 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
      841 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
      901 gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca
      961 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     1021 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     1081 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     1141 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     1201 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     1261 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     1321 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     1381 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     1441 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     1501 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     1561 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     1621 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     1681 agacccacct cccaaccccg aggggaccca gagagggcct atttcccatg attccttcat
     1741 atttgcatat acgatacaag gctgttagag agataattag aattaatttg actgtaaaca
     1801 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     1861 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     1921 atttcttggc tttatatatc ttgtggaaag gacgaaacac cggttcactg ccgtataggc
     1981 agctaagaaa tgagacggca gccagggatc ctatccgtct cagttcactg ccgtataggc
     2041 agctaagaaa actttttttg aattctagat cttgagacaa atggcagtat tcatccacaa
     2101 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat
     2161 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg
     2221 ggtttattac agggacagca gagatccact ttggcgccgg ctcgagtggc tccggtgccc
     2281 gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca
     2341 attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact
     2401 ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga
     2461 acgttctttt tcgcaacggg tttgccgcca gaacacaggt gtcgtgacgc gggatcctag
     2521 cggcagcggc gccaccaact tcagcctgct gaagcaggcc ggcgacgtgg aggagaaccc
     2581 cggccccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag
     2641 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
     2701 tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
     2761 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
     2821 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
     2881 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
     2941 caaggagccc gcgtggttcc tggccaccgt cggagtctcg cccgaccacc agggcaaggg
     3001 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
     3061 cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
     3121 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
     3181 tgcctgaacg cgttaagtcg acaatcaacc tctggattac aaaatttgtg aaagattgac
     3241 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     3301 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     3361 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
     3421 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
     3481 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
     3541 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc
     3601 atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
     3661 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
     3721 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
     3781 cgcctccccg cgtcgacttt aagaccaatg acttacaagg cagctgtaga tcttagccac
     3841 tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg
     3901 ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc
     3961 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
     4021 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
     4081 tggaaaatct ctagcagtac gtatagtagt tcatgtcatc ttattattca gtatttataa
     4141 cttgcaaaga aatgaatatc agagagtgag aggaacttgt ttattgcagc ttataatggt
     4201 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4261 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggctcta gctatcccgc
     4321 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     4381 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     4441 agaagtagtg aggaggcttt tttggaggcc tagggacgta cccaattcgc cctatagtga
     4501 gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     4561 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     4621 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc
     4681 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     4741 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     4801 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     4861 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     4921 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4981 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     5041 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     5101 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     5161 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     5221 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     5281 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     5341 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     5401 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     5461 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     5521 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     5581 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     5641 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     5701 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     5761 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     5821 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     5881 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     5941 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     6001 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6061 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     6121 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     6181 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     6241 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     6301 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     6361 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     6421 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     6481 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     6541 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     6601 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     6661 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     6721 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     6781 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     6841 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     6901 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     6961 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     7021 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     7081 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     7141 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
     7201 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     7261 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa
     7321 agggaacaaa agctggagct gcaagcttaa
//
