LOCUS       sequence-215176-        3025 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Single short guide RNA targeting CAATGTATCTTATCATGTC.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3025)
  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 3025)
  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: sg3
FEATURES             Location/Qualifiers
     source          1..3025
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(289..1080,1081..1149))
                     /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     912..931
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1150..1254)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1391..1639
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1391..1411
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1562..1581
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1756..1780)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1808..1824)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1808..1824)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1821..1843)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1832..1848
                     /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(1856..1862,1863..1880,1881..1886))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1901..1922
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        1981..2176
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2027..2162
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2182..2316
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2219..2238)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2273..2292
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2384..2401)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(2555..3025,1..118))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2635..2654)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
       61 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
      121 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
      181 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
      241 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
      301 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
      361 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
      421 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
      481 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
      541 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
      601 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
      661 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
      721 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
      781 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
      841 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
      901 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
      961 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     1021 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     1081 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     1141 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     1201 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     1261 tccccgaaaa gtgccacctg gtcgacattg attattgact agtaagaatt ctgcccccgg
     1321 gtgtacaaaa aagcaggctt taaaggaacc aattcagtcg actggatccg gtaccaaggt
     1381 cgggcaggaa gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc
     1441 tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac
     1501 gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
     1561 ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt
     1621 gtggaaagga cgaaacaccg caatgtatct tatcatgtcg tttcagagct atgctggaaa
     1681 cagcatagca agttgaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
     1741 ggtgcttttt tctagaccca gctttcttgt acaaagttgg cattacgcca agcttggcgt
     1801 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     1861 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     1921 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagcggatcc
     1981 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     2041 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     2101 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     2161 cctaggcttt tgcaaaaagc taacttgttt attgcagctt ataatggtta caaataaagc
     2221 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     2281 tccaaactca tcaatgtatc ttatcatgtc tggatccgct gcattaatga atcggccaac
     2341 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     2401 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     2461 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     2521 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     2581 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     2641 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     2701 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct
     2761 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     2821 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     2881 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     2941 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     3001 tatttggtat ctgcgctctg ctgaa
//
