LOCUS       sequence-222974-        3026 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 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: sg2
FEATURES             Location/Qualifiers
     source          1..3026
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        59..307
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     59..79
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     230..249
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(424..448)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(486..502)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(486..502)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(499..521)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    510..526
                     /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(534..540,541..558,559..564))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    579..600
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        659..854
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      705..840
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    860..994
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(897..916)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     951..970
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1062..1079)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1233..1821)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1313..1332)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1992..2783,2784..2852))
                     /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     2615..2634
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2853..2957)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gcaggcttta aaggaaccaa ttcagtcgac tggatccggt accaaggtcg ggcaggaaga
       61 gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat
      121 aattagaatt aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa
      181 gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat
      241 gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg
      301 aaacaccgaa cctctacaaa tgtggtagtt tcagagctat gctggaaaca gcatagcaag
      361 ttgaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttc
      421 tagacccagc tttcttgtac aaagttggca ttatgccccc gggcgccaag cttggcgtaa
      481 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
      541 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
      601 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc
      661 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
      721 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
      781 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
      841 taggcttttg caaaaagcta acttgtttat tgcagcttat aatggttaca aataaagcaa
      901 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
      961 caaactcatc aatgtatctt atcatgtctg gatccgctgc attaatgaat cggccaacgc
     1021 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     1081 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     1141 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     1201 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     1261 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     1321 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     1381 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     1441 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     1501 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     1561 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     1621 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     1681 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     1741 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     1801 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     1861 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     1921 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     1981 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     2041 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     2101 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     2161 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     2221 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     2281 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     2341 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     2401 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     2461 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     2521 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     2581 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     2641 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     2701 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     2761 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     2821 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     2881 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     2941 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgggtcgac attgattatt
     3001 gactagtaag aattctgtac aaaaaa
//
