LOCUS       sequence-232549-        5831 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway Donor; dCasRx ORF; No promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5831)
  AUTHORS   Du M, Jillette N, Zhu JJ, Li S, Cheng AW
  TITLE     CRISPR artificial splicing factors.
  JOURNAL   Nat Commun. 2020 Jun 12;11(1):2973. doi: 10.1038/s41467-020-16806-4.
  PUBMED    32532987
REFERENCE   2  (bases 1 to 5831)
  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: "2020-06-12"; volume: "11(1):2973. doi"; pages: " 
            10.1038/s41467-020-16806-4"
COMMENT     Sequence Label: pAC1822_pCR8-dCasRx
FEATURES             Location/Qualifiers
     source          1..5831
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(157..184)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(276..362)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     411..433
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     425..442
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     426..442
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    458..557
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             587..607
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             617..3514
                     /codon_start=1
                     /product="RNA-targeting Type VI CRISPR protein from 
                     Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
                     2018)"
                     /label=RfxCas13d
                     /translation="IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSI
                     RSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEK
                     RYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTVY
                     TYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIIN
                     YGNECYDILALLSGLAHWVVANNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNE
                     LTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKD
                     MSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVI
                     NLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAG
                     RDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFF
                     KDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDEN
                     GNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQK
                     KQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGREN
                     AEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEE
                     KGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKA
                     EEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFANKAVALEVARYVHA
                     YINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVP
                     FGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS"
     CDS             3524..3544
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3554..3580
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(3599..3698)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3715..3734)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3716..3734)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3739..3755)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3739..3755)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4186..4977
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      5070..5658
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5559..5578
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5812..5829
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
       61 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatac
      121 gcgtaccgct agccaggaag agtttgtaga aacgcaaaaa ggccatccgt caggatggcc
      181 ttctgcttag tttgatgcct ggcagtttat ggcgggcgtc ctgcccgcca ccctccgggc
      241 cgttgcttca caacgatcaa atccgctccc ggcggatttg tcctactcag gagagcgttc
      301 accgacaaac aacagataaa acgaaaggcc cagtcttccg actgagcctt tcgttttatt
      361 tgatgcctgg cagttcccta ctctcgcgtt aacgctagca tggatgtttt cccagtcacg
      421 acgttgtaaa acgacggcca gtcttaagct cgggccccaa ataatgattt tattttgact
      481 gatagtgacc tgttcgttgc aacaaattga tgagcaatgc ttttttataa tgccaacttt
      541 gtacaaaaaa gcaggctccg aattcaccgg tgccgccacc atgagcccca agaagaagag
      601 aaaggtggag gccagcatcg aaaaaaaaaa gtccttcgcc aagggcatgg gcgtgaagtc
      661 cacactcgtg tccggctcca aagtgtacat gacaaccttc gccgaaggca gcgacgccag
      721 gctggaaaag atcgtggagg gcgacagcat caggagcgtg aatgagggcg aggccttcag
      781 cgctgaaatg gccgataaaa acgccggcta taagatcggc aacgccaaat tcagccatcc
      841 taagggctac gccgtggtgg ctaacaaccc tctgtataca ggacccgtcc agcaggatat
      901 gctcggcctg aaggaaactc tggaaaagag gtacttcggc gagagcgctg atggcaatga
      961 caatatttgt atccaggtga tccataacat cctggacatt gaaaaaatcc tcgccgaata
     1021 cattaccaac gccgcctacg ccgtcaacaa tatctccggc ctggataagg acattattgg
     1081 attcggcaag ttctccacag tgtataccta cgacgaattc aaagaccccg agcaccatag
     1141 ggccgctttc aacaataacg ataagctcat caacgccatc aaggcccagt atgacgagtt
     1201 cgacaacttc ctcgataacc ccagactcgg ctatttcggc caggcctttt tcagcaagga
     1261 gggcagaaat tacatcatca attacggcaa cgaatgctat gacattctgg ccctcctgag
     1321 cggactggcg cactgggtgg tcgctaacaa cgaagaagag tccaggatct ccaggacctg
     1381 gctctacaac ctcgataaga acctcgacaa cgaatacatc tccaccctca actacctcta
     1441 cgacaggatc accaatgagc tgaccaactc cttctccaag aactccgccg ccaacgtgaa
     1501 ctatattgcc gaaactctgg gaatcaaccc tgccgaattc gccgaacaat atttcagatt
     1561 cagcattatg aaagagcaga aaaacctcgg attcaatatc accaagctca gggaagtgat
     1621 gctggacagg aaggatatgt ccgagatcag gaaaaatcat aaggtgttcg actccatcag
     1681 gaccaaggtc tacaccatga tggactttgt gatttatagg tattacatcg aagaggatgc
     1741 caaggtggct gccgccaata agtccctccc cgataatgag aagtccctga gcgagaagga
     1801 tatctttgtg attaacctga ggggctcctt caacgacgac cagaaggatg ccctctacta
     1861 cgatgaagct aatagaattt ggagaaagct cgaaaatatc atgcacaaca tcaaggaatt
     1921 taggggaaac aagacaagag agtataagaa gaaggacgcc cctagactgc ccagaatcct
     1981 gcccgctggc cgtgatgttt ccgccttcag caaactcatg tatgccctga ccatgttcct
     2041 ggatggcaag gagatcaacg acctcctgac caccctgatt aataaattcg ataacatcca
     2101 gagcttcctg aaggtgatgc ctctcatcgg agtcaacgct aagttcgtgg aggaatacgc
     2161 ctttttcaaa gactccgcca agatcgccga tgagctgagg ctgatcaagt ccttcgctag
     2221 aatgggagaa cctattgccg atgccaggag ggccatgtat atcgacgcca tccgtatttt
     2281 aggaaccaac ctgtcctatg atgagctcaa ggccctcgcc gacacctttt ccctggacga
     2341 gaacggaaac aagctcaaga aaggcaagca cggcatgaga aatttcatta ttaataacgt
     2401 gatcagcaat aaaaggttcc actacctgat cagatacggt gatcctgccc acctccatga
     2461 gatcgccaaa aacgaggccg tggtgaagtt cgtgctcggc aggatcgctg acatccagaa
     2521 aaaacagggc cagaacggca agaaccagat cgacaggtac tacgaaactt gtatcggaaa
     2581 ggataagggc aagagcgtga gcgaaaaggt ggacgctctc acaaagatca tcaccggaat
     2641 gaactacgac caattcgaca agaaaaggag cgtcattgag gacaccggca gggaaaacgc
     2701 cgagagggag aagtttaaaa agatcatcag cctgtacctc accgtgatct accacatcct
     2761 caagaatatt gtcaatatca acgccaggta cgtcatcgga ttccattgcg tcgagcgtga
     2821 tgctcaactg tacaaggaga aaggctacga catcaatctc aagaaactgg aagagaaggg
     2881 attcagctcc gtcaccaagc tctgcgctgg cattgatgaa actgcccccg ataagagaaa
     2941 ggacgtggaa aaggagatgg ctgaaagagc caaggagagc attgacagcc tcgagagcgc
     3001 caaccccaag ctgtatgcca attacatcaa atacagcgac gagaagaaag ccgaggagtt
     3061 caccaggcag attaacaggg agaaggccaa aaccgccctg aacgcctacc tgaggaacac
     3121 caagtggaat gtgatcatca gggaggacct cctgagaatt gacaacaaga catgtaccct
     3181 gttcgcaaac aaggccgtcg ccctggaagt ggccaggtat gtccacgcct atatcaacga
     3241 cattgccgag gtcaattcct acttccaact gtaccattac atcatgcaga gaattatcat
     3301 gaatgagagg tacgagaaaa gcagcggaaa ggtgtccgag tacttcgacg ctgtgaatga
     3361 cgagaagaag tacaacgata ggctcctgaa actgctgtgt gtgcctttcg gctactgtat
     3421 ccccaggttt aagaacctga gcatcgaggc cctgttcgat aggaacgagg ccgccaagtt
     3481 cgacaaggag aaaaagaagg tgtccggcaa ttccggatcc ggacctaaga aaaagaggaa
     3541 ggtggcggcc gcttacccat acgatgttcc agattacgct tgattaatta agaattcgac
     3601 ccagctttct tgtacaaagt tggcattata aaaaataatt gctcatcaat ttgttgcaac
     3661 gaacaggtca ctatcagtca aaataaaatc attatttgcc atccagctga tatcccctat
     3721 agtgagtcgt attacatggt catagctgtt tcctggcagc tctggcccgt gtctcaaaat
     3781 ctctgatgtt acattgcaca agataaaaat atatcatcat gcctcctcta gaccagccag
     3841 gacagaaatg cctcgacttc gctgctaccc aaggttgccg ggtgacgcac accgtggaaa
     3901 cggatgaagg cacgaaccca gtggacataa gcctgttcgg ttcgtaagct gtaatgcaag
     3961 tagcgtatgc gctcacgcaa ctggtccaga accttgaccg aacgcagcgg tggtaacggc
     4021 gcagtggcgg ttttcatggc ttgttatgac tgtttttttg gggtacagtc tatgcctcgg
     4081 gcatccaagc agcaagcgcg ttacgccgtg ggtcgatgtt tgatgttatg gagcagcaac
     4141 gatgttacgc agcagggcag tcgccctaaa acaaagttaa acattatgag ggaagcggtg
     4201 atcgccgaag tatcgactca actatcagag gtagttggcg tcatcgagcg ccatctcgaa
     4261 ccgacgttgc tggccgtaca tttgtacggc tccgcagtgg atggcggcct gaagccacac
     4321 agtgatattg atttgctggt tacggtgacc gtaaggcttg atgaaacaac gcggcgagct
     4381 ttgatcaacg accttttgga aacttcggct tcccctggag agagcgagat tctccgcgct
     4441 gtagaagtca ccattgttgt gcacgacgac atcattccgt ggcgttatcc agctaagcgc
     4501 gaactgcaat ttggagaatg gcagcgcaat gacattcttg caggtatctt cgagccagcc
     4561 acgatcgaca ttgatctggc tatcttgctg acaaaagcaa gagaacatag cgttgccttg
     4621 gtaggtccag cggcggagga actctttgat ccggttcctg aacaggatct atttgaggcg
     4681 ctaaatgaaa ccttaacgct atggaactcg ccgcccgact gggctggcga tgagcgaaat
     4741 gtagtgctta cgttgtcccg catttggtac agcgcagtaa ccggcaaaat cgcgccgaag
     4801 gatgtcgctg ccgactgggc aatggagcgc ctgccggccc agtatcagcc cgtcatactt
     4861 gaagctagac aggcttatct tggacaagaa gaagatcgct tggcctcgcg cgcagatcag
     4921 ttggaagaat ttgtccacta cgtgaaaggc gagatcacca aggtagtcgg caaataaccc
     4981 tcgagccacc catgaccaaa atcccttaac gtgagttacg cgtcgttcca ctgagcgtca
     5041 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     5101 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     5161 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     5221 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     5281 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     5341 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     5401 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     5461 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     5521 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     5581 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     5641 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     5701 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     5761 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     5821 gtgagcgagg a
//
