LOCUS       sequence-266706-        7895 bp    DNA     circular SYN 14-APR-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 7895)
  TITLE     Unpublished
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7895)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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            "Unpublished"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_Puro
FEATURES             Location/Qualifiers
     source          1..7895
                     /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..4926
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4327..4346)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4823..4843
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4955..4972)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4961..5168
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5177..5317
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5177..5306
                     /label=AAV2 ITR
     rep_origin      5392..5847
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5479..5498)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5689..5710
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5864..5883)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5983..6005
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6043..6061)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6129..6233
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6234..6302,6303..7094)
                     /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(6452..6471)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7265..7853
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7754..7773
                     /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 ggcccaatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg
     4381 gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat
     4441 ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg
     4501 ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg ccgtggcggt ctggaccacg
     4561 ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg
     4621 agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc
     4681 aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc ccgaccacca gggcaagggt
     4741 ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc
     4801 ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc
     4861 accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt
     4921 gcctgacgta cgctaactag agctcgctga tcagcctcga ctgtgccttc tagttgccag
     4981 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     5041 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     5101 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagagaa tagcaggcat
     5161 gctggggagc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     5221 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     5281 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc
     5341 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     5401 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     5461 ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     5521 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     5581 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     5641 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     5701 tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta taagggattt
     5761 tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     5821 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     5881 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     5941 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     6001 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     6061 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     6121 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     6181 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     6241 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     6301 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     6361 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     6421 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     6481 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     6541 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     6601 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     6661 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     6721 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     6781 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     6841 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     6901 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta
     6961 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     7021 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     7081 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     7141 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     7201 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     7261 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     7321 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     7381 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     7441 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     7501 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     7561 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     7621 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     7681 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     7741 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     7801 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     7861 gcaacgcggc ctttttacgg ttcctggcct tttgc
//
