LOCUS       sequence-222998-        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"
     protein_bind    complement(150..174)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        259..507
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     protein_bind    complement(624..648)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(676..692)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(676..692)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(689..711)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    700..716
                     /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(724..730,731..748,749..754))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    769..790
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        849..1044
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      895..1030
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1050..1184
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1087..1106)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1141..1160
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1252..1269)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1423..2011)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1503..1522)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2182..2973,2974..3042))
                     /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     2805..2824
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3043..3147)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3266..3514
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     protein_bind    complement(3631..3655)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        join(3738..3953,1..33)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
ORIGIN
        1 gctttatata tcttgtggaa aggacgaaac accgaacctc tacaaatgtg gtagtttcag
       61 agctatgctg gaaacagcat agcaagttga aataaggcta gtccgttatc aacttgaaaa
      121 agtggcaccg agtcggtgct tttttctaga cccagctttc ttgtacaaag ttggcattat
      181 gcccccgggt gtacaaaaaa gcaggcttta aaggaaccaa ttcagtcgac tggatccggt
      241 accaaggtcg ggcaggaaga gggcctattt cccatgattc cttcatattt gcatatacga
      301 tacaaggctg ttagagagat aattagaatt aatttgactg taaacacaaa gatattagta
      361 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
      421 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta
      481 tatatcttgt ggaaaggacg aaacaccgca atgtatctta tcatgtcgtt tcagagctat
      541 gctggaaaca gcatagcaag ttgaaataag gctagtccgt tatcaacttg aaaaagtggc
      601 accgagtcgg tgcttttttc tagacccagc tttcttgtac aaagttggca ttacgccaag
      661 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
      721 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
      781 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
      841 gcggatccgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
      901 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
      961 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     1021 tttggaggcc taggcttttg caaaaagcta acttgtttat tgcagcttat aatggttaca
     1081 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     1141 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatccgctgc attaatgaat
     1201 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     1261 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     1321 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     1381 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     1441 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     1501 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     1561 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     1621 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     1681 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     1741 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     1801 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     1861 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     1921 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     1981 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     2041 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     2101 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     2161 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     2221 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     2281 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     2341 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     2401 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     2461 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     2521 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     2581 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     2641 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     2701 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     2761 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     2821 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     2881 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     2941 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     3001 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     3061 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     3121 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgggtcgac
     3181 attgattatt gactagttgt acaaaaaagc aggtttaaag gaaccaattc agtcgactgg
     3241 atccggtacc aaggtcgggc aggaagaggg cctatttccc atgattcctt catatttgca
     3301 tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat
     3361 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
     3421 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
     3481 ggctttatat atcttatgga aaggacgaaa caccggtata gcatacatta tacggtttca
     3541 gagctatgct ggaaacagca tagcaagttg aaataaggct agtccgttat caacttgaaa
     3601 aagtggcacc gagtcggtgc ttttttctag acccagcttt cttgtacaaa gttggcatta
     3661 aagaattctg tacaaaaaag caggctttaa aggaaccaat tcagtcgact ggatccggta
     3721 ccaaggtcgg gcaggaagag ggcctatttc ccatgattcc ttcatatttg catatacgat
     3781 acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag atattagtac
     3841 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     3901 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttg
//
