LOCUS       sequence-215177-        3953 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Triple short guide RNA targeting GTATAGCATACATTATACG, 
            TACCACATTTGTAGAGGTT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3953)
  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 3953)
  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: sg1+sg2+sg3
FEATURES             Location/Qualifiers
     source          1..3953
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        196..444
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     protein_bind    complement(561..585)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        668..916
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     protein_bind    complement(1033..1057)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        1142..1390
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     protein_bind    complement(1507..1531)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1559..1575)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1559..1575)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1572..1594)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1583..1599
                     /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(1607..1613,1614..1631,1632..1637))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1652..1673
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        1732..1927
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      1778..1913
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1933..2067
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1970..1989)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2024..2043
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2135..2152)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2306..2894)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2386..2405)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3065..3856,3857..3925))
                     /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     3688..3707
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(3926..3953,1..77))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
       61 caaatagggg ttccgcgcac atttccccga aaagtgccac ctggtcgaca ttgattattg
      121 actagttgta caaaaaagca ggctttaaag gaaccaattc agtcgactgg atccggtacc
      181 aaggtcgggc aggaagaggg cctatttccc atgattcctt catatttgca tatacgatac
      241 aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa
      301 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
      361 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
      421 atcttgtgga aaggacgaaa caccggtata gcatacatta tacggtttca gagctatgct
      481 ggaaacagca tagcaagttg aaataaggct agtccgttat caacttgaaa aagtggcacc
      541 gagtcggtgc ttttttctag acccagcttt cttgtacaaa gttggcatta aagaattctg
      601 tacaaaaaag caggctttaa aggaaccaat tcagtcgact ggatccggta ccaaggtcgg
      661 gcaggaagag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
      721 tagagagata attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
      781 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
      841 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg
      901 gaaaggacga aacaccgaac ctctacaaat gtggtagttt cagagctatg ctggaaacag
      961 catagcaagt tgaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
     1021 gcttttttct agacccagct ttcttgtaca aagttggcat tatgcccccg ggtgtacaaa
     1081 aaagcaggct ttaaaggaac caattcagtc gactggatcc ggtaccaagg tcgggcagga
     1141 agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
     1201 gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
     1261 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     1321 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
     1381 acgaaacacc gcaatgtatc ttatcatgtc gtttcagagc tatgctggaa acagcatagc
     1441 aagttgaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
     1501 ttctagaccc agctttcttg tacaaagttg gcattacgcc aagcttggcg taatcatggt
     1561 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     1621 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     1681 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc cgcatctcaa
     1741 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     1801 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     1861 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     1921 ttgcaaaaag ctaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     1981 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     2041 atcaatgtat cttatcatgt ctggatccgc tgcattaatg aatcggccaa cgcgcgggga
     2101 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     2161 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     2221 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     2281 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     2341 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     2401 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     2461 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc
     2521 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     2581 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     2641 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     2701 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     2761 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     2821 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     2881 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     2941 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     3001 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     3061 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     3121 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     3181 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     3241 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     3301 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     3361 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     3421 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     3481 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     3541 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3601 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     3661 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     3721 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     3781 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     3841 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     3901 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agc
//
