LOCUS       sequence-337350-        7358 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for encoding a human codon-optimized 
            minidCas13X.1-REPAIRv2 driven by CMV promoter,EGFP driven by CMV 
            promoter and U6-driven crRNAs cloning site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7358)
  AUTHORS   Xu C, Zhou Y, Xiao Q, He B, Geng G, Wang Z, Cao B, Dong X, Bai W, 
            Wang Y, Wang X, Zhou D, Yuan T, Huo X, Lai J, Yang H
  TITLE     Programmable RNA editing with compact CRISPR-Cas13 systems from 
            uncultivated microbes.
  JOURNAL   Nat Methods. 2021 May;18(5):499-506. doi: 
            10.1038/s41592-021-01124-4. Epub 2021 May 3.
  PUBMED    33941935
REFERENCE   2  (bases 1 to 7358)
  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"; doi: 
            "10.1038/s41592-021-01124-4"; journalName: "Nat Methods"; date: 
            "2021-05"; volume: "18"; issue: "5"; pages: "499-506"
COMMENT     Sequence Label: 
            U6-BbsI-DR_CMV-minidCas13X.1-REPAIRv2-BGHpA_CMV-EGFP-BGHpA
FEATURES             Location/Qualifiers
     source          1..7358
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        14..118
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(119..187,188..979)
                     /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(337..356)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1150..1738
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1639..1658
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        1832..2080
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1832..1852
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2003..2022
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        2171..2550
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2551..2754
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     intron          2846..2978
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     CDS             3011..3031
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4364..4384
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5603..5629
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(5657..5674)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5663..5870
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        5879..6182
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6183..6386
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             join(6415..6417,6418..6420,6421..7134)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(6415..6417,6418..6420,6421..7131)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6460..6481)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6721..6740)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7068..7089
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    join(7141..7358,1..7)
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 tctatggaag cttcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
       61 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
      121 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
      181 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
      241 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
      301 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
      361 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
      421 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
      481 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
      541 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
      601 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
      661 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
      721 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
      781 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
      841 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
      901 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
      961 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     1021 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     1081 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     1141 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     1201 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     1261 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     1321 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     1381 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     1441 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     1501 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     1561 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     1621 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     1681 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     1741 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     1801 ctttcctgcg ttatcccctg attctctcga ggagggccta tttcccatga ttccttcata
     1861 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
     1921 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     1981 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     2041 tttcttggct ttatatatct tgtggaaagg acgaaacacc ggggtcttcg atattcaagc
     2101 gtcggaagac ctgctggagc agcccccgat ttgtggggtg attacagctt tttttgtgga
     2161 cggtgatatc ggcattgatt attgactagt tattaatagt aatcaattac ggggtcatta
     2221 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     2281 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     2341 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     2401 gcagtacatc aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa
     2461 tggcccgcct ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac
     2521 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg
     2581 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
     2641 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg
     2701 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag aggtcgttta
     2761 gtgaaccgtc agatcactag tagctttatt gcggtagttt atcacagtta aattgctaac
     2821 gcagtcagtg ctcgactgat cacaggtaag tatcaaggtt acaagacagg tttaaggagg
     2881 ccaatagaaa ctgggcttgt cgagacagag aagattcttg cgtttctgat aggcacctat
     2941 tggtcttact gacatccact ttgcctttct ctccacaggg gtaccgaagc cgctagtcga
     3001 cgccaccatg cccaagaaga agcggaaggt gctgagcatg tactgcctga aggacagcag
     3061 attcaccaag gcctgggata agcgggtgct gctgttcaga gacatcctgg cccagctggg
     3121 aagaatcccc gccgaggcct acgagtacta ccacggcgag cagggtgata agaagagagc
     3181 taacgacaat gagggcacaa atcccaagcg gcacaaggac aagttcatcg aatttgcact
     3241 gcactacctg gaagcccagc acagcgagat ctgcttcggc agacgccaca tcgtgcggga
     3301 agaggccggc gccggcgatg agcacaagaa gcaccggacc aagggaaagg tggtggtgga
     3361 cttcagcaag aaggacgagg accagagcta ctatatctcc aagaacaacg tgatcgtgcg
     3421 gatcgacaag aacgccggcc ctagaagcta ccggatgggc ctgaacgagc tgaagtacct
     3481 cgtgctgctg agcctgcagg ggaagggcga cgatgccatc gccaagctgt acagatacag
     3541 acagcacgtg gagaacatcc tggatgtggt gaaggtgacc gataaggata accacgtgtt
     3601 cctgccccgc ttcgtgctgg agcagcacgg catcggcaga aaggccttca agcagcggat
     3661 cgatggacgg gtgaagcacg tgcggggcgt gtgggagaag aagaaggccg ccaccaatga
     3721 aatgaccctg cacgagaagg ccagagacat cctgcagtac gtgaacgaaa actgcacccg
     3781 gtccttcaac cctggcgaat acaacagact gctggtgtgc ctggtgggca aggacgtgga
     3841 gaactttcag gccggcctga agcggctgca gctggccgaa aggatcgatg gccgggtgta
     3901 ctccatcttc gcccagacca gcaccatcaa tgagatgcac caggtggtgt gcgaccagat
     3961 cctgaaccgg ctgtgcagaa tcggcgacca gaagctgtac gattacgtgg gactgggcaa
     4021 gaaggacgaa atcgactaca agcagaaggt ggcctggttc aaggagcaca tcagcatccg
     4081 gagaggattc ctgagaaaga agttctggta cgatagcaag aagggattcg caaagctggt
     4141 ggaggaacac ctggagtccg gcggcggcca gcgcgacgtg ggcctggaca agaagtacta
     4201 ccacatcgac gccatcggca gattcgaggg cgccaacccc gccctgtacg agaccctggc
     4261 cagagatcgg ctgtgcctca tgatggccca gtacttcctg ggcagcgtga gaaaggaact
     4321 gggcaacaag attgtgtgga gcaacgacag catcgaactg cctcccaaga agaagcggaa
     4381 ggtgggtgga ggcggaggtt ctgggggagg aggtagtggc ggtggtggtt caggaggcgg
     4441 cggaagccag ctgcatttac cgcaggtttt agctgacgct gtctcacgcc tggtcctggg
     4501 taagtttggt gacctgaccg acaacttctc ctcccctcac gctcgcagaa aagtgctggc
     4561 tggagtcgtc atgacaacag gcacagatgt taaagatgcc aaggtgataa gtgtttctac
     4621 aggaggcaaa tgtattaatg gtgaatacat gagtgatcgt ggccttgcat taaatgactg
     4681 ccatgcagaa ataatatctc ggagatcctt gctcagattt ctttatacac aacttgagct
     4741 ttacttaaat aacaaagatg atcaaaaaag atccatcttt cagaaatcag agcgaggggg
     4801 gtttaggctg aaggagaatg tccagtttca tctgtacatc agcacctctc cctgtggaga
     4861 tgccagaatc ttctcaccac atgagccaat cctggaagaa ccagcagata gacacccaaa
     4921 tcgtaaagca agaggacagc tacggaccaa aatagagtct ggtcagggga cgattccagt
     4981 gcgctccaat gcgagcatcc aaacgtggga cggggtgctg caaggggagc ggctgctcac
     5041 catgtcctgc agtgacaaga ttgcacgctg gaacgtggtg ggcatccagg gatcactgct
     5101 cagcattttc gtggagccca tttacttctc gagcatcatc ctgggcagcc tttaccacgg
     5161 ggaccacctt tccagggcca tgtaccagcg gatctccaac atagaggacc tgccacctct
     5221 ctacaccctc aacaagcctt tgctcagtgg catcagcaat gcagaagcac ggcagccagg
     5281 gaaggccccc aacttcagtg tcaactggac ggtaggcgac tccgctattg aggtcatcaa
     5341 cgccacgact gggaaggatg agctgggccg cgcgtcccgc ctgtgtaagc acgcgttgta
     5401 ctgtcgctgg atgcgtgtgc acggcaaggt tccctcccac ttactacgct ccaagattac
     5461 caagcccaac gtgtaccatg agtccaagct ggcggcaaag gagtaccagg ccgccaaggc
     5521 gcgtctgttc acagccttca tcaaggcggg gctgggggcc tgggtggaga agcccaccga
     5581 gcaggaccag ttctcactca cgtacccata cgacgtacca gattacgctt aagaattcct
     5641 agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     5701 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     5761 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     5821 caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga gcggccgccg
     5881 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     5941 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     6001 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     6061 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     6121 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     6181 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
     6241 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
     6301 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
     6361 ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc tagcatggtg
     6421 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     6481 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     6541 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     6601 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     6661 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     6721 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     6781 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     6841 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     6901 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     6961 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     7021 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     7081 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaggatcc
     7141 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     7201 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     7261 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     7321 gggaaaacaa tagcaggcat gctggggatg cggtgggc
//
