LOCUS       sequence-215175-        3025 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Single short guide RNA targeting TACCACATTTGTAGAGGTT.
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: sg2
FEATURES             Location/Qualifiers
     source          1..3025
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(294..1085,1086..1154))
                     /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     917..936
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1155..1259)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1386..1634
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1386..1406
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1557..1576
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1751..1775)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1813..1829)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1813..1829)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1826..1848)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1837..1853
                     /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(1861..1867,1868..1885,1886..1891))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1906..1927
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        1986..2181
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2032..2167
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2187..2321
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2224..2243)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2278..2297
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2389..2406)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(2560..3025,1..123))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2640..2659)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
       61 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
      121 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
      181 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
      241 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
      301 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
      361 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
      421 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
      481 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
      541 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
      601 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
      661 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
      721 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
      781 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
      841 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
      901 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
      961 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     1021 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     1081 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     1141 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     1201 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     1261 acatttcccc gaaaagtgcc acctggtcga cattgattat tgactagtaa gaattctgta
     1321 caaaaaagca ggctttaaag gaaccaattc agtcgactgg atccggtacc aaggtcgggc
     1381 aggaagaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
     1441 gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
     1501 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
     1561 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
     1621 aaggacgaaa caccgaacct ctacaaatgt ggtagtttca gagctatgct ggaaacagca
     1681 tagcaagttg aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     1741 ttttttctag acccagcttt cttgtacaaa gttggcatta tgcccccggg cgccaagctt
     1801 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     1861 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     1921 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg
     1981 gatccgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     2041 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     2101 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     2161 ggaggcctag gcttttgcaa aaagctaact tgtttattgc agcttataat ggttacaaat
     2221 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     2281 gtttgtccaa actcatcaat gtatcttatc atgtctggat ccgctgcatt aatgaatcgg
     2341 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     2401 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     2461 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     2521 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     2581 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     2641 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     2701 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc
     2761 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     2821 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     2881 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     2941 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     3001 gacagtattt ggtatctgcg ctctg
//
