LOCUS       sequence-232523-        6666 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient transfection; Expresses dCasRx-FKBP; CAGGS promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6666)
  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 6666)
  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: pAC1807_pmax-dCasRx-FKBP
FEATURES             Location/Qualifiers
     source          1..6666
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        140..519
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        520..723
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     674..694
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          815..947
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     protein_bind    962..986
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1004..1013
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1013..1078
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1103..1123
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1127..1147
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1151..1171
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1253..1273
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1283..4180
                     /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             4190..4210
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4220..4246
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4358..4681
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     protein_bind    complement(4697..4721)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4774..4815
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(4841..4860)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    4870..4951
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     4895..4914
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(5149..5737)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5229..5248)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5818..6612)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(5818..6612)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5929..5948)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6539..6558
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 tgtcaatatt ggccattagc catattattc attggttata tagcataaat caatattggc
       61 tattggccat tgcatacgtt gtatctatat cataatatgt acatttatat tggctcatgt
      121 ccaatatgac cgccatgttg gcattgatta ttgactagtt attaatagta atcaattacg
      181 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
      241 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
      301 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
      361 gcccacttgg cagtacatca agtgtatcat atgccaagtc cgccccctat tgacgtcaat
      421 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttacggga ctttcctact
      481 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
      541 accaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
      601 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaataac
      661 cccgccccgt tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
      721 ggtcgtttag tgaaccgtca gatcactagt agctttattg cggtagttta tcacagttaa
      781 attgctaacg cagtcagtgc tcgactgatc acaggtaagt atcaaggtta caagacaggt
      841 ttaaggaggc caatagaaac tgggcttgtc gagacagaga agattcttgc gtttctgata
      901 ggcacctatt ggtcttactg acatccactt tgcctttctc tccacaggga aaaaacaatt
      961 gacaagtttg tacaaaaaag caggctccga attcaccggt gccgccacca tggactacaa
     1021 ggatcacgac ggtgactata aggatcatga catcgactat aaggacgatg acgataagat
     1081 cgatggcgga ggcggatctg atccaaaaaa gaagagaaag gtagatccaa aaaagaagag
     1141 aaaggtagat ccaaaaaaga agagaaaggt aggatctacc ggatctagaa acgatggtgg
     1201 tggtggaagc gggggtgggg gcagcggtgg agggggaagc gggcgcgcca gccccaagaa
     1261 gaagagaaag gtggaggcca gcatcgaaaa aaaaaagtcc ttcgccaagg gcatgggcgt
     1321 gaagtccaca ctcgtgtccg gctccaaagt gtacatgaca accttcgccg aaggcagcga
     1381 cgccaggctg gaaaagatcg tggagggcga cagcatcagg agcgtgaatg agggcgaggc
     1441 cttcagcgct gaaatggccg ataaaaacgc cggctataag atcggcaacg ccaaattcag
     1501 ccatcctaag ggctacgccg tggtggctaa caaccctctg tatacaggac ccgtccagca
     1561 ggatatgctc ggcctgaagg aaactctgga aaagaggtac ttcggcgaga gcgctgatgg
     1621 caatgacaat atttgtatcc aggtgatcca taacatcctg gacattgaaa aaatcctcgc
     1681 cgaatacatt accaacgccg cctacgccgt caacaatatc tccggcctgg ataaggacat
     1741 tattggattc ggcaagttct ccacagtgta tacctacgac gaattcaaag accccgagca
     1801 ccatagggcc gctttcaaca ataacgataa gctcatcaac gccatcaagg cccagtatga
     1861 cgagttcgac aacttcctcg ataaccccag actcggctat ttcggccagg cctttttcag
     1921 caaggagggc agaaattaca tcatcaatta cggcaacgaa tgctatgaca ttctggccct
     1981 cctgagcgga ctggcgcact gggtggtcgc taacaacgaa gaagagtcca ggatctccag
     2041 gacctggctc tacaacctcg ataagaacct cgacaacgaa tacatctcca ccctcaacta
     2101 cctctacgac aggatcacca atgagctgac caactccttc tccaagaact ccgccgccaa
     2161 cgtgaactat attgccgaaa ctctgggaat caaccctgcc gaattcgccg aacaatattt
     2221 cagattcagc attatgaaag agcagaaaaa cctcggattc aatatcacca agctcaggga
     2281 agtgatgctg gacaggaagg atatgtccga gatcaggaaa aatcataagg tgttcgactc
     2341 catcaggacc aaggtctaca ccatgatgga ctttgtgatt tataggtatt acatcgaaga
     2401 ggatgccaag gtggctgccg ccaataagtc cctccccgat aatgagaagt ccctgagcga
     2461 gaaggatatc tttgtgatta acctgagggg ctccttcaac gacgaccaga aggatgccct
     2521 ctactacgat gaagctaata gaatttggag aaagctcgaa aatatcatgc acaacatcaa
     2581 ggaatttagg ggaaacaaga caagagagta taagaagaag gacgccccta gactgcccag
     2641 aatcctgccc gctggccgtg atgtttccgc cttcagcaaa ctcatgtatg ccctgaccat
     2701 gttcctggat ggcaaggaga tcaacgacct cctgaccacc ctgattaata aattcgataa
     2761 catccagagc ttcctgaagg tgatgcctct catcggagtc aacgctaagt tcgtggagga
     2821 atacgccttt ttcaaagact ccgccaagat cgccgatgag ctgaggctga tcaagtcctt
     2881 cgctagaatg ggagaaccta ttgccgatgc caggagggcc atgtatatcg acgccatccg
     2941 tattttagga accaacctgt cctatgatga gctcaaggcc ctcgccgaca ccttttccct
     3001 ggacgagaac ggaaacaagc tcaagaaagg caagcacggc atgagaaatt tcattattaa
     3061 taacgtgatc agcaataaaa ggttccacta cctgatcaga tacggtgatc ctgcccacct
     3121 ccatgagatc gccaaaaacg aggccgtggt gaagttcgtg ctcggcagga tcgctgacat
     3181 ccagaaaaaa cagggccaga acggcaagaa ccagatcgac aggtactacg aaacttgtat
     3241 cggaaaggat aagggcaaga gcgtgagcga aaaggtggac gctctcacaa agatcatcac
     3301 cggaatgaac tacgaccaat tcgacaagaa aaggagcgtc attgaggaca ccggcaggga
     3361 aaacgccgag agggagaagt ttaaaaagat catcagcctg tacctcaccg tgatctacca
     3421 catcctcaag aatattgtca atatcaacgc caggtacgtc atcggattcc attgcgtcga
     3481 gcgtgatgct caactgtaca aggagaaagg ctacgacatc aatctcaaga aactggaaga
     3541 gaagggattc agctccgtca ccaagctctg cgctggcatt gatgaaactg cccccgataa
     3601 gagaaaggac gtggaaaagg agatggctga aagagccaag gagagcattg acagcctcga
     3661 gagcgccaac cccaagctgt atgccaatta catcaaatac agcgacgaga agaaagccga
     3721 ggagttcacc aggcagatta acagggagaa ggccaaaacc gccctgaacg cctacctgag
     3781 gaacaccaag tggaatgtga tcatcaggga ggacctcctg agaattgaca acaagacatg
     3841 taccctgttc gcaaacaagg ccgtcgccct ggaagtggcc aggtatgtcc acgcctatat
     3901 caacgacatt gccgaggtca attcctactt ccaactgtac cattacatca tgcagagaat
     3961 tatcatgaat gagaggtacg agaaaagcag cggaaaggtg tccgagtact tcgacgctgt
     4021 gaatgacgag aagaagtaca acgataggct cctgaaactg ctgtgtgtgc ctttcggcta
     4081 ctgtatcccc aggtttaaga acctgagcat cgaggccctg ttcgatagga acgaggccgc
     4141 caagttcgac aaggagaaaa agaaggtgtc cggcaattcc ggatccggac ctaagaaaaa
     4201 gaggaaggtg gcggccgctt acccatacga tgttccagat tacgctggcg gccgcggggg
     4261 aggcggttcc ggtggcggcg gaagcggagg tggaggatca gggccggccg gaggtggctc
     4321 atctggcgga ggtcagatct cgtacgcgtc ccggggcgga gtgcaggtgg aaaccatctc
     4381 cccaggagac gggcgcacct tccccaagcg cggccagacc tgcgtggtgc actacaccgg
     4441 gatgcttgaa gatggaaaga aatttgattc ctcccgggac agaaacaagc cctttaagtt
     4501 tatgctaggc aagcaggagg tgatccgagg ctgggaagaa ggggttgccc agatgagtgt
     4561 gggtcagaga gccaaactga ctatatctcc agattatgcc tatggtgcca ctgggcaccc
     4621 aggcatcatc ccaccacatg ccactctcgt cttcgatgtg gagcttctaa aactggaatg
     4681 attaattaag aattcgaccc agctttcttg tacaaagtgg ttgatatcca gcacagtggc
     4741 ggccgctcga gtctagaggg cccgcggttc gaaggtaagc ctatccctaa ccctctcctc
     4801 ggtctcgatt ctacgcgtac cggttagcaa ttgttttttc gatgagtttg gacaaaccac
     4861 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     4921 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     4981 tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg
     5041 tacttaagag ggggagacca aagggcgaga cgttaaggcc tcacgtgaca tgtgagcaaa
     5101 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     5161 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     5221 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     5281 gaccctgccg cttacgggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     5341 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5401 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5461 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5521 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5581 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5641 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5701 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5761 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgccgtctca
     5821 gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc
     5881 gtaaagcacg aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt
     5941 agccaacgct atgtcctgat agcggtccgc cacacccagc cggccacagt cgatgaatcc
     6001 agaaaagcgg ccattttcca ccatgatatt cggcaagcag gcatcgccat gggtcacgac
     6061 gagatcctcg ccgtcgggca tgctcgcctt gagcctggcg aacagttcgg ctggcgcgag
     6121 cccctgatgc tcttcgtcca gatcatcctg atcgacaaga ccggcttcca tccgagtacg
     6181 tgctctctcg atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt
     6241 atgcagccgc cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtgaga
     6301 tgacaggaga tcctgccccg gcacttcgcc caatagcagc cagtcccttc ccgcttcagt
     6361 gacaacgtcg agtacagctg cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc
     6421 tgcctcgtct tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg
     6481 gcgcccctgc gctgacagcc ggaacacggc ggcatcagag cagccgattg tctgttgtgc
     6541 ccagtcatag ccgaatagcc tctccaccca agcggccgga gaacctgcgt gcaatccatc
     6601 ttgttcaatc ataatattat tgaagcattt atcagggttc gtctcgtccc ggtctcctcc
     6661 catgca
//
