LOCUS       sequence-331088-        2957 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6-driven expression of human VEGFA targeting presgRNA compatible 
            with CasRx..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2957)
  AUTHORS   Chuang YF, Wang PY, Kumar S, Lama S, Lin FL, Liu GS
  TITLE     Methods for in vitro CRISPR/CasRx-Mediated RNA Editing.
  JOURNAL   Front Cell Dev Biol. 2021 Jun 11;9:667879. doi: 
            10.3389/fcell.2021.667879. eCollection 2021.
  PUBMED    34178991
REFERENCE   2  (bases 1 to 2957)
  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: "Front Cell 
            Dev Biol."; date: "2021-06-11"; volume: "9:667879. doi"; pages: " 
            10.3389/fcell.2021.667879. eCollection 2021"
COMMENT     Sequence Label: pM116: CasRx VEGFA presgRNA
FEATURES             Location/Qualifiers
     source          1..2957
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        11..259
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     11..31
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     182..201
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        261..296
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        327..362
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     rep_origin      444..899
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(531..550)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     741..762
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(916..935)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1035..1057
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1095..1113)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1181..1285
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1286..1354,1355..2146)
                     /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(1504..1523)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2317..2905
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2806..2825
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctcagctagc gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc
       61 tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac
      121 gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
      181 ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt
      241 gtggaaagga cgaaacaccg caagtaaacc cctaccaact ggtcggggtt tgaaactatg
      301 tgctggcctt ggtgaggttt gatccgcaag taaaccccta ccaactggtc ggggtttgaa
      361 actttttttg aattctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
      421 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
      481 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc
      541 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
      601 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
      661 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
      721 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat
      781 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa
      841 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt
      901 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
      961 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     1021 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     1081 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     1141 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     1201 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     1261 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     1321 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     1381 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     1441 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     1501 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     1561 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     1621 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     1681 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     1741 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     1801 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     1861 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     1921 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     1981 atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa
     2041 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     2101 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     2161 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     2221 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     2281 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     2341 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     2401 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     2461 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     2521 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     2581 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     2641 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     2701 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     2761 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     2821 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     2881 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     2941 cttttgctgg ccttttg
//
