LOCUS       sequence-222983-        3026 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Single short guide RNA targeting GTATAGCATACATTATACG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3026)
  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 3026)
  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
FEATURES             Location/Qualifiers
     source          1..3026
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        64..312
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     64..84
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     235..254
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(429..453)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(499..515)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(499..515)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(512..534)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    523..539
                     /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(547..553,554..571,572..577))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    592..613
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        672..867
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      718..853
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    873..1007
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(910..929)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     964..983
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1075..1092)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1246..1834)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1326..1345)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2005..2796,2797..2865))
                     /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     2628..2647
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2866..2970)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaaaagcagg ctttaaagga accaattcag tcgactggat ccggtaccaa ggtcgggcag
       61 gaagagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
      121 gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
      181 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
      241 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
      301 ggacgaaaca ccggtatagc atacattata cggtttcaga gctatgctgg aaacagcata
      361 gcaagttgaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
      421 ttttctagac ccagctttct tgtacaaagt tggcattaaa gaattctgcc cccgggcgcc
      481 aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
      541 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
      601 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
      661 ccagcggatc cgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
      721 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
      781 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
      841 ttttttggag gcctaggctt ttgcaaaaag ctaacttgtt tattgcagct tataatggtt
      901 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
      961 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatccgc tgcattaatg
     1021 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     1081 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     1141 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     1201 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     1261 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     1321 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     1381 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     1441 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     1501 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     1561 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     1621 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     1681 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     1741 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     1801 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     1861 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     1921 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     1981 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     2041 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     2101 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     2161 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     2221 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     2281 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     2341 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     2401 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     2461 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     2521 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     2581 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     2641 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     2701 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     2761 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     2821 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     2881 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     2941 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgggtc
     3001 gacattgatt attgactagt tgtaca
//
