LOCUS       sequence-222972-        3490 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Double short guide RNA targeting TACCACATTTGTAGAGGTT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3490)
  AUTHORS   Rui Y, Varanasi M, Mendes S, Yamagata HM, Wilson DR, Green JJ
  TITLE     Poly(Beta-Amino Ester) Nanoparticles Enable Nonviral Delivery of 
            CRISPR-Cas9 Plasmids for Gene Knockout and Gene Deletion.
  JOURNAL   Mol Ther Nucleic Acids. 2020 Apr 21;20:661-672. doi: 
            10.1016/j.omtn.2020.04.005.
  PUBMED    32380416
REFERENCE   2  (bases 1 to 3490)
  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 
            Nucleic Acids."; date: "2020-04-21"; volume: "20:661-672. doi"; 
            pages: " 10.1016/j.omtn.2020.04.005"
COMMENT     Sequence Label: sg2+sg3
FEATURES             Location/Qualifiers
     source          1..3490
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        80..328
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     protein_bind    complement(445..469)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        554..802
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     protein_bind    complement(919..943)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(971..987)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(971..987)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(984..1006)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    995..1011
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(1019..1025,1026..1043,1044..1049))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1064..1085
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        1144..1339
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      1190..1325
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1345..1479
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1382..1401)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1436..1455
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1547..1564)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1718..2306)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1798..1817)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2477..3268,3269..3337))
                     /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     3100..3119
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3338..3442)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtaagaattc tgtacaaaaa agcaggcttt aaaggaacca attcagtcga ctggatccgg
       61 taccaaggtc gggcaggaag agggcctatt tcccatgatt ccttcatatt tgcatatacg
      121 atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa agatattagt
      181 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
      241 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
      301 atatatcttg tggaaaggac gaaacaccga acctctacaa atgtggtagt ttcagagcta
      361 tgctggaaac agcatagcaa gttgaaataa ggctagtccg ttatcaactt gaaaaagtgg
      421 caccgagtcg gtgctttttt ctagacccag ctttcttgta caaagttggc attatgcccc
      481 cgggtgtaca aaaaagcagg ctttaaagga accaattcag tcgactggat ccggtaccaa
      541 ggtcgggcag gaagagggcc tatttcccat gattccttca tatttgcata tacgatacaa
      601 ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa
      661 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
      721 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
      781 cttgtggaaa ggacgaaaca ccgcaatgta tcttatcatg tcgtttcaga gctatgctgg
      841 aaacagcata gcaagttgaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
      901 gtcggtgctt ttttctagac ccagctttct tgtacaaagt tggcattacg ccaagcttgg
      961 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     1021 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     1081 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagcgga
     1141 tccgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     1201 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     1261 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     1321 aggcctaggc ttttgcaaaa agctaacttg tttattgcag cttataatgg ttacaaataa
     1381 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     1441 ttgtccaaac tcatcaatgt atcttatcat gtctggatcc gctgcattaa tgaatcggcc
     1501 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     1561 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     1621 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     1681 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     1741 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     1801 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     1861 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     1921 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     1981 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     2041 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     2101 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     2161 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     2221 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     2281 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     2341 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     2401 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     2461 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     2521 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     2581 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     2641 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     2701 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     2761 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     2821 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     2881 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     2941 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     3001 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     3061 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     3121 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     3181 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     3241 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     3301 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     3361 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     3421 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctggg tcgacattga
     3481 ttattgacta
//
