LOCUS       sequence-259132-        8006 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All in one overexpression of CasRx and guideRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8006)
  TITLE     Unpublished
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8006)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pAK_DR30_CasRx_mCherry
FEATURES             Location/Qualifiers
     source          1..8006
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        20..268
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     20..40
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     191..210
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        270..299
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     enhancer        448..733
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        735..1012
                     /label=chicken beta-actin promoter
     intron          1013..1240
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1264..1284
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1294..4191
                     /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
                     YGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNE
                     LTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKD
                     MSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVI
                     NLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAG
                     RDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFF
                     KDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDEN
                     GNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQK
                     KQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGREN
                     AEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEE
                     KGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKA
                     EEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHA
                     YINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVP
                     FGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS"
     CDS             4201..4221
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4231..4257
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4273..4326
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             4327..5037
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(4472..4490)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(5066..5083)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5072..5279
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5288..5428
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5288..5417
                     /label=AAV2 ITR
     rep_origin      5503..5958
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5590..5609)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5800..5821
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5975..5994)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6094..6116
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6154..6172)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6240..6344
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6345..6413,6414..7205)
                     /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(6563..6582)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7376..7964
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7865..7884
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tggccttttg ctcacatgtg agggcctatt tcccatgatt ccttcatatt tgcatatacg
       61 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt
      121 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
      181 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
      241 atatatcttg tggaaaggac gaaacaccga acccctacca actggtcggg gtttgaaacg
      301 ggtcttcgag aagacctttt ttttggacag cagagatcca gtttggttaa ttaatgcaaa
      361 gatggataaa gttttaaaca gagaggaatc tttgcagcta atggaccttc taggtcttga
      421 aaggagtggg aattctctag aggtacccgt tacataactt acggtaaatg gcccgcctgg
      481 ctgaccgccc aacgaccccc gcccattgac gtcaatagta acgccaatag ggactttcca
      541 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
      601 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcattg
      661 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
      721 cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc
      781 ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc
      841 gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc
      901 gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat
      961 ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc
     1021 tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct
     1081 ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg
     1141 taattagctg agcaagaggt aagggtttaa gggatggttg gttggtgggg tattaatgtt
     1201 taattacctg gagcacctgc ctgaaatcac tttttttcag gttggaccgg tgccaccatg
     1261 agccccaaga agaagagaaa ggtggaggcc agcatcgaaa aaaaaaagtc cttcgccaag
     1321 ggcatgggcg tgaagtccac actcgtgtcc ggctccaaag tgtacatgac aaccttcgcc
     1381 gaaggcagcg acgccaggct ggaaaagatc gtggagggcg acagcatcag gagcgtgaat
     1441 gagggcgagg ccttcagcgc tgaaatggcc gataaaaacg ccggctataa gatcggcaac
     1501 gccaaattca gccatcctaa gggctacgcc gtggtggcta acaaccctct gtatacagga
     1561 cccgtccagc aggatatgct cggcctgaag gaaactctgg aaaagaggta cttcggcgag
     1621 agcgctgatg gcaatgacaa tatttgtatc caggtgatcc ataacatcct ggacattgaa
     1681 aaaatcctcg ccgaatacat taccaacgcc gcctacgccg tcaacaatat ctccggcctg
     1741 gataaggaca ttattggatt cggcaagttc tccacagtgt atacctacga cgaattcaaa
     1801 gaccccgagc accatagggc cgctttcaac aataacgata agctcatcaa cgccatcaag
     1861 gcccagtatg acgagttcga caacttcctc gataacccca gactcggcta tttcggccag
     1921 gcctttttca gcaaggaggg cagaaattac atcatcaatt acggcaacga atgctatgac
     1981 attctggccc tcctgagcgg actgaggcac tgggtggtcc ataacaacga agaagagtcc
     2041 aggatctcca ggacctggct ctacaacctc gataagaacc tcgacaacga atacatctcc
     2101 accctcaact acctctacga caggatcacc aatgagctga ccaactcctt ctccaagaac
     2161 tccgccgcca acgtgaacta tattgccgaa actctgggaa tcaaccctgc cgaattcgcc
     2221 gaacaatatt tcagattcag cattatgaaa gagcagaaaa acctcggatt caatatcacc
     2281 aagctcaggg aagtgatgct ggacaggaag gatatgtccg agatcaggaa aaatcataag
     2341 gtgttcgact ccatcaggac caaggtctac accatgatgg actttgtgat ttataggtat
     2401 tacatcgaag aggatgccaa ggtggctgcc gccaataagt ccctccccga taatgagaag
     2461 tccctgagcg agaaggatat ctttgtgatt aacctgaggg gctccttcaa cgacgaccag
     2521 aaggatgccc tctactacga tgaagctaat agaatttgga gaaagctcga aaatatcatg
     2581 cacaacatca aggaatttag gggaaacaag acaagagagt ataagaagaa ggacgcccct
     2641 agactgccca gaatcctgcc cgctggccgt gatgtttccg ccttcagcaa actcatgtat
     2701 gccctgacca tgttcctgga tggcaaggag atcaacgacc tcctgaccac cctgattaat
     2761 aaattcgata acatccagag cttcctgaag gtgatgcctc tcatcggagt caacgctaag
     2821 ttcgtggagg aatacgcctt tttcaaagac tccgccaaga tcgccgatga gctgaggctg
     2881 atcaagtcct tcgctagaat gggagaacct attgccgatg ccaggagggc catgtatatc
     2941 gacgccatcc gtattttagg aaccaacctg tcctatgatg agctcaaggc cctcgccgac
     3001 accttttccc tggacgagaa cggaaacaag ctcaagaaag gcaagcacgg catgagaaat
     3061 ttcattatta ataacgtgat cagcaataaa aggttccact acctgatcag atacggtgat
     3121 cctgcccacc tccatgagat cgccaaaaac gaggccgtgg tgaagttcgt gctcggcagg
     3181 atcgctgaca tccagaaaaa acagggccag aacggcaaga accagatcga caggtactac
     3241 gaaacttgta tcggaaagga taagggcaag agcgtgagcg aaaaggtgga cgctctcaca
     3301 aagatcatca ccggaatgaa ctacgaccaa ttcgacaaga aaaggagcgt cattgaggac
     3361 accggcaggg aaaacgccga gagggagaag tttaaaaaga tcatcagcct gtacctcacc
     3421 gtgatctacc acatcctcaa gaatattgtc aatatcaacg ccaggtacgt catcggattc
     3481 cattgcgtcg agcgtgatgc tcaactgtac aaggagaaag gctacgacat caatctcaag
     3541 aaactggaag agaagggatt cagctccgtc accaagctct gcgctggcat tgatgaaact
     3601 gcccccgata agagaaagga cgtggaaaag gagatggctg aaagagccaa ggagagcatt
     3661 gacagcctcg agagcgccaa ccccaagctg tatgccaatt acatcaaata cagcgacgag
     3721 aagaaagccg aggagttcac caggcagatt aacagggaga aggccaaaac cgccctgaac
     3781 gcctacctga ggaacaccaa gtggaatgtg atcatcaggg aggacctcct gagaattgac
     3841 aacaagacat gtaccctgtt cagaaacaag gccgtccacc tggaagtggc caggtatgtc
     3901 cacgcctata tcaacgacat tgccgaggtc aattcctact tccaactgta ccattacatc
     3961 atgcagagaa ttatcatgaa tgagaggtac gagaaaagca gcggaaaggt gtccgagtac
     4021 ttcgacgctg tgaatgacga gaagaagtac aacgataggc tcctgaaact gctgtgtgtg
     4081 cctttcggct actgtatccc caggtttaag aacctgagca tcgaggccct gttcgatagg
     4141 aacgaggccg ccaagttcga caaggagaaa aagaaggtgt ccggcaattc cggatccgga
     4201 cctaagaaaa agaggaaggt ggcggccgct tacccatacg atgttccaga ttacgctgct
     4261 agcggcagtg gagagggcag aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct
     4321 ggcccaatgg tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc
     4381 ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc
     4441 gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc
     4501 ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg
     4561 aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg
     4621 gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg
     4681 caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc
     4741 cccgtaatgc agaagaaaac catgggctgg gaggcctcct ccgagcggat gtaccccgag
     4801 gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac
     4861 gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac
     4921 aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag
     4981 tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtgacgt
     5041 acgctaacta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     5101 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     5161 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     5221 ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca tgctggggag
     5281 cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
     5341 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg
     5401 agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat
     5461 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg
     5521 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct
     5581 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     5641 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     5701 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     5761 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     5821 gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt
     5881 cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     5941 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     6001 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     6061 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     6121 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     6181 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     6241 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     6301 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     6361 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     6421 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     6481 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     6541 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     6601 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     6661 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     6721 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     6781 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     6841 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     6901 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     6961 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     7021 gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag
     7081 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     7141 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     7201 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     7261 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     7321 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     7381 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     7441 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     7501 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     7561 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     7621 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     7681 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     7741 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     7801 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     7861 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     7921 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     7981 cctttttacg gttcctggcc ttttgc
//
