LOCUS       sequence-232447-        6675 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient transfection; Expresses FKBP-dCasRx; CAGGS promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6675)
  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 6675)
  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: pAC1806_pmax-FKBP-dCasRx
FEATURES             Location/Qualifiers
     source          1..6675
                     /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             1061..1381
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             1412..1432
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1436..1456
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1460..1480
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1562..1582
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1592..4489
                     /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             4499..4519
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4529..4555
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4622..4687
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    complement(4706..4730)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4783..4824
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(4850..4869)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    4879..4960
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     4904..4923
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(5158..5746)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5238..5257)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5827..6621)
                     /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(5827..6621)
                     /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(5938..5957)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6548..6567
                     /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 tgggaggtgg
     1021 ctcatctggc ggaggtcaga tctcgtacgc gtcccggggc ggagtgcagg tggaaaccat
     1081 ctccccagga gacgggcgca ccttccccaa gcgcggccag acctgcgtgg tgcactacac
     1141 cgggatgctt gaagatggaa agaaatttga ttcctcccgg gacagaaaca agccctttaa
     1201 gtttatgcta ggcaagcagg aggtgatccg aggctgggaa gaaggggttg cccagatgag
     1261 tgtgggtcag agagccaaac tgactatatc tccagattat gcctatggtg ccactgggca
     1321 cccaggcatc atcccaccac atgccactct cgtcttcgat gtggagcttc taaaactgga
     1381 aaacgttatc gatggcggag gcggatctga tccaaaaaag aagagaaagg tagatccaaa
     1441 aaagaagaga aaggtagatc caaaaaagaa gagaaaggta ggatctaccg gatctagaaa
     1501 cgatggtggt ggtggaagcg ggggtggggg cagcggtgga gggggaagcg ggcgcgccag
     1561 ccccaagaag aagagaaagg tggaggccag catcgaaaaa aaaaagtcct tcgccaaggg
     1621 catgggcgtg aagtccacac tcgtgtccgg ctccaaagtg tacatgacaa ccttcgccga
     1681 aggcagcgac gccaggctgg aaaagatcgt ggagggcgac agcatcagga gcgtgaatga
     1741 gggcgaggcc ttcagcgctg aaatggccga taaaaacgcc ggctataaga tcggcaacgc
     1801 caaattcagc catcctaagg gctacgccgt ggtggctaac aaccctctgt atacaggacc
     1861 cgtccagcag gatatgctcg gcctgaagga aactctggaa aagaggtact tcggcgagag
     1921 cgctgatggc aatgacaata tttgtatcca ggtgatccat aacatcctgg acattgaaaa
     1981 aatcctcgcc gaatacatta ccaacgccgc ctacgccgtc aacaatatct ccggcctgga
     2041 taaggacatt attggattcg gcaagttctc cacagtgtat acctacgacg aattcaaaga
     2101 ccccgagcac catagggccg ctttcaacaa taacgataag ctcatcaacg ccatcaaggc
     2161 ccagtatgac gagttcgaca acttcctcga taaccccaga ctcggctatt tcggccaggc
     2221 ctttttcagc aaggagggca gaaattacat catcaattac ggcaacgaat gctatgacat
     2281 tctggccctc ctgagcggac tggcgcactg ggtggtcgct aacaacgaag aagagtccag
     2341 gatctccagg acctggctct acaacctcga taagaacctc gacaacgaat acatctccac
     2401 cctcaactac ctctacgaca ggatcaccaa tgagctgacc aactccttct ccaagaactc
     2461 cgccgccaac gtgaactata ttgccgaaac tctgggaatc aaccctgccg aattcgccga
     2521 acaatatttc agattcagca ttatgaaaga gcagaaaaac ctcggattca atatcaccaa
     2581 gctcagggaa gtgatgctgg acaggaagga tatgtccgag atcaggaaaa atcataaggt
     2641 gttcgactcc atcaggacca aggtctacac catgatggac tttgtgattt ataggtatta
     2701 catcgaagag gatgccaagg tggctgccgc caataagtcc ctccccgata atgagaagtc
     2761 cctgagcgag aaggatatct ttgtgattaa cctgaggggc tccttcaacg acgaccagaa
     2821 ggatgccctc tactacgatg aagctaatag aatttggaga aagctcgaaa atatcatgca
     2881 caacatcaag gaatttaggg gaaacaagac aagagagtat aagaagaagg acgcccctag
     2941 actgcccaga atcctgcccg ctggccgtga tgtttccgcc ttcagcaaac tcatgtatgc
     3001 cctgaccatg ttcctggatg gcaaggagat caacgacctc ctgaccaccc tgattaataa
     3061 attcgataac atccagagct tcctgaaggt gatgcctctc atcggagtca acgctaagtt
     3121 cgtggaggaa tacgcctttt tcaaagactc cgccaagatc gccgatgagc tgaggctgat
     3181 caagtccttc gctagaatgg gagaacctat tgccgatgcc aggagggcca tgtatatcga
     3241 cgccatccgt attttaggaa ccaacctgtc ctatgatgag ctcaaggccc tcgccgacac
     3301 cttttccctg gacgagaacg gaaacaagct caagaaaggc aagcacggca tgagaaattt
     3361 cattattaat aacgtgatca gcaataaaag gttccactac ctgatcagat acggtgatcc
     3421 tgcccacctc catgagatcg ccaaaaacga ggccgtggtg aagttcgtgc tcggcaggat
     3481 cgctgacatc cagaaaaaac agggccagaa cggcaagaac cagatcgaca ggtactacga
     3541 aacttgtatc ggaaaggata agggcaagag cgtgagcgaa aaggtggacg ctctcacaaa
     3601 gatcatcacc ggaatgaact acgaccaatt cgacaagaaa aggagcgtca ttgaggacac
     3661 cggcagggaa aacgccgaga gggagaagtt taaaaagatc atcagcctgt acctcaccgt
     3721 gatctaccac atcctcaaga atattgtcaa tatcaacgcc aggtacgtca tcggattcca
     3781 ttgcgtcgag cgtgatgctc aactgtacaa ggagaaaggc tacgacatca atctcaagaa
     3841 actggaagag aagggattca gctccgtcac caagctctgc gctggcattg atgaaactgc
     3901 ccccgataag agaaaggacg tggaaaagga gatggctgaa agagccaagg agagcattga
     3961 cagcctcgag agcgccaacc ccaagctgta tgccaattac atcaaataca gcgacgagaa
     4021 gaaagccgag gagttcacca ggcagattaa cagggagaag gccaaaaccg ccctgaacgc
     4081 ctacctgagg aacaccaagt ggaatgtgat catcagggag gacctcctga gaattgacaa
     4141 caagacatgt accctgttcg caaacaaggc cgtcgccctg gaagtggcca ggtatgtcca
     4201 cgcctatatc aacgacattg ccgaggtcaa ttcctacttc caactgtacc attacatcat
     4261 gcagagaatt atcatgaatg agaggtacga gaaaagcagc ggaaaggtgt ccgagtactt
     4321 cgacgctgtg aatgacgaga agaagtacaa cgataggctc ctgaaactgc tgtgtgtgcc
     4381 tttcggctac tgtatcccca ggtttaagaa cctgagcatc gaggccctgt tcgataggaa
     4441 cgaggccgcc aagttcgaca aggagaaaaa gaaggtgtcc ggcaattccg gatccggacc
     4501 taagaaaaag aggaaggtgg cggccgctta cccatacgat gttccagatt acgctggcgg
     4561 ccgcggggga ggcggttccg gtggcggcgg aagcggaggt ggaggatcag ggccggccat
     4621 ggactacaag gatcacgacg gtgactataa ggatcatgac atcgactata aggacgatga
     4681 cgataagtga ttaattaaga attcgaccca gctttcttgt acaaagtggt tgatatccag
     4741 cacagtggcg gccgctcgag tctagagggc ccgcggttcg aaggtaagcc tatccctaac
     4801 cctctcctcg gtctcgattc tacgcgtacc ggttagcaat tgttttttcg atgagtttgg
     4861 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     4921 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca
     4981 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta
     5041 caaatgtggt acttaagagg gggagaccaa agggcgagac gttaaggcct cacgtgacat
     5101 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5161 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5221 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5281 tcctgttccg accctgccgc ttacgggata cctgtccgcc tttctccctt cgggaagcgt
     5341 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5401 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5461 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5521 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5581 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5641 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5701 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5761 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5821 gccgtctcag aagaactcgt caagaaggcg atagaaggcg atgcgctgcg aatcgggagc
     5881 ggcgataccg taaagcacga ggaagcggtc agcccattcg ccgccaagct cttcagcaat
     5941 atcacgggta gccaacgcta tgtcctgata gcggtccgcc acacccagcc ggccacagtc
     6001 gatgaatcca gaaaagcggc cattttccac catgatattc ggcaagcagg catcgccatg
     6061 ggtcacgacg agatcctcgc cgtcgggcat gctcgccttg agcctggcga acagttcggc
     6121 tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac cggcttccat
     6181 ccgagtacgt gctctctcga tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg
     6241 atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct cggcaggagc
     6301 aaggtgagat gacaggagat cctgccccgg cacttcgccc aatagcagcc agtcccttcc
     6361 cgcttcagtg acaacgtcga gtacagctgc gcaaggaacg cccgtcgtgg ccagccacga
     6421 tagccgcgct gcctcgtctt gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa
     6481 aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc agccgattgt
     6541 ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag aacctgcgtg
     6601 caatccatct tgttcaatca taatattatt gaagcattta tcagggttcg tctcgtcccg
     6661 gtctcctccc atgca
//
