LOCUS       sequence-215178-        3489 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 3489)
  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 3489)
  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..3489
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(296..1087,1088..1156))
                     /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     919..938
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1157..1261)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1388..1636
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     protein_bind    complement(1753..1777)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        1862..2110
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     protein_bind    complement(2227..2251)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2279..2295)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2279..2295)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2292..2314)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2303..2319
                     /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(2327..2333,2334..2351,2352..2357))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2372..2393
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2452..2647
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2498..2633
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2653..2787
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2690..2709)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2744..2763
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2855..2872)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(3026..3489,1..125))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3106..3125)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
       61 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
      121 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
      181 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
      241 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
      301 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
      361 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
      421 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
      481 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
      541 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
      601 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
      661 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
      721 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
      781 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
      841 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
      901 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
      961 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     1021 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     1081 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     1141 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     1201 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     1261 gcacatttcc ccgaaaagtg ccacctggtc gacattgatt attgactagt aagaattctg
     1321 tacaaaaaag caggctttaa aggaaccaat tcagtcgact ggatccggta ccaaggtcgg
     1381 gcaggaagag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
     1441 tagagagata attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
     1501 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
     1561 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg
     1621 gaaaggacga aacaccgaac ctctacaaat gtggtagttt cagagctatg ctggaaacag
     1681 catagcaagt tgaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
     1741 gcttttttct agacccagct ttcttgtaca aagttggcat tatgcccccg ggtgtacaaa
     1801 aaagcaggct ttaaaggaac caattcagtc gactggatcc ggtaccaagg tcgggcagga
     1861 agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
     1921 gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
     1981 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     2041 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
     2101 acgaaacacc gcaatgtatc ttatcatgtc gtttcagagc tatgctggaa acagcatagc
     2161 aagttgaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
     2221 ttctagaccc agctttcttg tacaaagttg gcattacgcc aagcttggcg taatcatggt
     2281 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     2341 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     2401 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc cgcatctcaa
     2461 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     2521 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     2581 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     2641 ttgcaaaaag ctaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     2701 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     2761 atcaatgtat cttatcatgt ctggatccgc tgcattaatg aatcggccaa cgcgcgggga
     2821 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     2881 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     2941 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3001 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     3061 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     3121 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     3181 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc
     3241 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     3301 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     3361 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     3421 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     3481 tctgcgctc
//
