LOCUS       sequence-222973-        3026 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 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: sg3
FEATURES             Location/Qualifiers
     source          1..3026
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        49..297
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     49..69
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     220..239
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(414..438)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(466..482)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(466..482)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(479..501)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    490..506
                     /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(514..520,521..538,539..544))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    559..580
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        639..834
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      685..820
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    840..974
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(877..896)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     931..950
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1042..1059)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1213..1801)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1293..1312)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1972..2763,2764..2832))
                     /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     2595..2614
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2833..2937)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaggaaccaa ttcagtcgac tggatccggt accaaggtcg ggcaggaaga gggcctattt
       61 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattagaatt
      121 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt
      181 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta
      241 acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgca
      301 atgtatctta tcatgtcgtt tcagagctat gctggaaaca gcatagcaag ttgaaataag
      361 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttc tagacccagc
      421 tttcttgtac aaagttggca ttacgccaag cttggcgtaa tcatggtcat agctgtttcc
      481 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
      541 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
      601 cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta gtcagcaacc
      661 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
      721 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
      781 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagcta
      841 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
      901 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
      961 atcatgtctg gatccgctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     1021 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     1081 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     1141 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     1201 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     1261 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     1321 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     1381 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     1441 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     1501 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     1561 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     1621 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     1681 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     1741 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     1801 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     1861 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     1921 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     1981 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     2041 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     2101 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     2161 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     2221 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     2281 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     2341 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     2401 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     2461 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     2521 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     2581 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     2641 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     2701 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     2761 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     2821 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     2881 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     2941 atttccccga aaagtgccac ctgggtcgac attgattatt gactagtaag aattctgccc
     3001 ccgggtgtac aaaaaagcag gcttta
//
