LOCUS       sequence-463769-        7001 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To Express HA Tagged hfCas13d and mcherry from the mammalian EFS 
            promoter. The hfCas13d and mCherry are separated by a P2A site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7001)
  AUTHORS   Back F, Sandoval A, Vu LM, Hong VM, Bhaskara A, Rodriguez SR, 
            O'Brien JT, Kolber BJ, Kroener S, Ploski JE
  TITLE     Adeno-associated viral vector resource for the RNA-targeting Cas13d:
            A comparison of high-fidelity variants, DjCas13d and hfCas13d.
  JOURNAL   Mol Ther Methods Clin Dev. 2025 Aug 20;33(4):101565. doi: 
            10.1016/j.omtm.2025.101565. eCollection 2025 Dec 11.
  PUBMED    41020266
REFERENCE   2  (bases 1 to 7001)
  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: "Mol Ther 
            Methods Clin Dev."; date: "2025-08-20"; volume: "33(4):101565. doi";
            pages: " 10.1016/j.omtm.2025.101565. eCollection 2025 Dec 11"
COMMENT     Sequence Label: pAAV-EFS-hfCas13d-P2A-mCherry-pA
FEATURES             Location/Qualifiers
     source          1..7001
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   651..780
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        834..1045
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             1078..1098
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1108..4005
                     /codon_start=1
                     /product="RNA-targeting Type VI CRISPR protein from 
                     Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
                     2018)"
                     /label=RfxCas13d
                     /translation="IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSI
                     RSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEK
                     RYFGESADGNDNICIQVIHNILDIEKILVEYITNVVYVVNNISGLDKDIIGFGKFSTVY
                     TYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIIN
                     YGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNE
                     LTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKD
                     MSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVI
                     NLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAG
                     RDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFF
                     KDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDEN
                     GNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQK
                     KQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGREN
                     AEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEE
                     KGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKA
                     EEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHA
                     YINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVP
                     FGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS"
     CDS             4015..4035
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4045..4071
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4087..4143
                     /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             4144..4851
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(4286..4304)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4554..4573
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4858..4906
                     /note="synthetic polyadenylation signal"
     repeat_region   4914..5054
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4914..5043
                     /label=AAV2 ITR
     rep_origin      5129..5584
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5216..5235)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5426..5447
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5601..5620)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5720..5742
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5780..5798)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5866..5970
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5971..6039,6040..6831)
                     /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(6189..6208)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
      661 gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg
      721 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
      781 gcggcctcta gagctaggcg tcctagagtc gagtggctcc ggtgcccgtc agtgggcaga
      841 gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaacgggtgc
      901 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt
      961 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg
     1021 caacgggttt gccgccagaa cacaggtgtc gtgacgcgga ccggtgccac catgagcccc
     1081 aagaagaaga gaaaggtgga ggccagcatc gaaaaaaaaa agtccttcgc caagggcatg
     1141 ggcgtgaagt ccacactcgt gtccggctcc aaagtgtaca tgacaacctt cgccgaaggc
     1201 agcgacgcca ggctggaaaa gatcgtggag ggcgacagca tcaggagcgt gaatgagggc
     1261 gaggccttca gcgctgaaat ggccgataaa aacgccggct ataagatcgg caacgccaaa
     1321 ttcagccatc ctaagggcta cgccgtggtg gctaacaacc ctctgtatac aggacccgtc
     1381 cagcaggata tgctcggcct gaaggaaact ctggaaaaga ggtacttcgg cgagagcgct
     1441 gatggcaatg acaatatttg tatccaggtg atccataaca tcctggacat tgaaaaaatc
     1501 ctcgtggaat acattaccaa cgtggtgtac gtggtcaaca atatctccgg cctggataag
     1561 gacattattg gattcggcaa gttctccaca gtgtatacct acgacgaatt caaagacccc
     1621 gagcaccata gggccgcttt caacaataac gataagctca tcaacgccat caaggcccag
     1681 tatgacgagt tcgacaactt cctcgataac cccagactcg gctatttcgg ccaggccttt
     1741 ttcagcaagg agggcagaaa ttacatcatc aattacggca acgaatgcta tgacattctg
     1801 gccctcctga gcggactgag gcactgggtg gtccataaca acgaagaaga gtccaggatc
     1861 tccaggacct ggctctacaa cctcgataag aacctcgaca acgaatacat ctccaccctc
     1921 aactacctct acgacaggat caccaatgag ctgaccaact ccttctccaa gaactccgcc
     1981 gccaacgtga actatattgc cgaaactctg ggaatcaacc ctgccgaatt cgccgaacaa
     2041 tatttcagat tcagcattat gaaagagcag aaaaacctcg gattcaatat caccaagctc
     2101 agggaagtga tgctggacag gaaggatatg tccgagatca ggaaaaatca taaggtgttc
     2161 gactccatca ggaccaaggt ctacaccatg atggactttg tgatttatag gtattacatc
     2221 gaagaggatg ccaaggtggc tgccgccaat aagtccctcc ccgataatga gaagtccctg
     2281 agcgagaagg atatctttgt gattaacctg aggggctcct tcaacgacga ccagaaggat
     2341 gccctctact acgatgaagc taatagaatt tggagaaagc tcgaaaatat catgcacaac
     2401 atcaaggaat ttaggggaaa caagacaaga gagtataaga agaaggacgc ccctagactg
     2461 cccagaatcc tgcccgctgg ccgtgatgtt tccgccttca gcaaactcat gtatgccctg
     2521 accatgttcc tggatggcaa ggagatcaac gacctcctga ccaccctgat taataaattc
     2581 gataacatcc agagcttcct gaaggtgatg cctctcatcg gagtcaacgc taagttcgtg
     2641 gaggaatacg cctttttcaa agactccgcc aagatcgccg atgagctgag gctgatcaag
     2701 tccttcgcta gaatgggaga acctattgcc gatgccagga gggccatgta tatcgacgcc
     2761 atccgtattt taggaaccaa cctgtcctat gatgagctca aggccctcgc cgacaccttt
     2821 tccctggacg agaacggaaa caagctcaag aaaggcaagc acggcatgag aaatttcatt
     2881 attaataacg tgatcagcaa taaaaggttc cactacctga tcagatacgg tgatcctgcc
     2941 cacctccatg agatcgccaa aaacgaggcc gtggtgaagt tcgtgctcgg caggatcgct
     3001 gacatccaga aaaaacaggg ccagaacggc aagaaccaga tcgacaggta ctacgaaact
     3061 tgtatcggaa aggataaggg caagagcgtg agcgaaaagg tggacgctct cacaaagatc
     3121 atcaccggaa tgaactacga ccaattcgac aagaaaagga gcgtcattga ggacaccggc
     3181 agggaaaacg ccgagaggga gaagtttaaa aagatcatca gcctgtacct caccgtgatc
     3241 taccacatcc tcaagaatat tgtcaatatc aacgccaggt acgtcatcgg attccattgc
     3301 gtcgagcgtg atgctcaact gtacaaggag aaaggctacg acatcaatct caagaaactg
     3361 gaagagaagg gattcagctc cgtcaccaag ctctgcgctg gcattgatga aactgccccc
     3421 gataagagaa aggacgtgga aaaggagatg gctgaaagag ccaaggagag cattgacagc
     3481 ctcgagagcg ccaaccccaa gctgtatgcc aattacatca aatacagcga cgagaagaaa
     3541 gccgaggagt tcaccaggca gattaacagg gagaaggcca aaaccgccct gaacgcctac
     3601 ctgaggaaca ccaagtggaa tgtgatcatc agggaggacc tcctgagaat tgacaacaag
     3661 acatgtaccc tgttcagaaa caaggccgtc cacctggaag tggccaggta tgtccacgcc
     3721 tatatcaacg acattgccga ggtcaattcc tacttccaac tgtaccatta catcatgcag
     3781 agaattatca tgaatgagag gtacgagaaa agcagcggaa aggtgtccga gtacttcgac
     3841 gctgtgaatg acgagaagaa gtacaacgat aggctcctga aactgctgtg tgtgcctttc
     3901 ggctactgta tccccaggtt taagaacctg agcatcgagg ccctgttcga taggaacgag
     3961 gccgccaagt tcgacaagga gaaaaagaag gtgtccggca attccggatc cggacctaag
     4021 aaaaagagga aggtggccgc tgcgtaccca tacgatgttc cagattacgc tgccagcggc
     4081 tccggagcca cgaacttctc tctgttaaag caagcaggag acgtggaaga aaaccccggt
     4141 cccgtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
     4201 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
     4261 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     4321 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
     4381 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
     4441 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac
     4501 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
     4561 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
     4621 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
     4681 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
     4741 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
     4801 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta aaagcttaat
     4861 aaaagatctt tattttcatt agatctgtgt gttggttttt tgtgtgcggc cgcaggaacc
     4921 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     4981 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     5041 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     5101 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     5161 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
     5221 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     5281 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     5341 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     5401 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     5461 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     5521 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     5581 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     5641 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     5701 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     5761 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     5821 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     5881 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     5941 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     6001 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     6061 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     6121 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     6181 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     6241 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     6301 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     6361 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     6421 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     6481 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     6541 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     6601 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     6661 gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat
     6721 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     6781 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     6841 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     6901 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     6961 cactgagcgt cagaccccgt agaaaagatc aaaggatctt c
//
