LOCUS       sequence-248422-        2919 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Single template U6 promoter minimal size plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2919)
  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 2919)
  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: PTG
FEATURES             Location/Qualifiers
     source          1..2919
                     /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        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(1645..1669)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1707..1723)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1707..1723)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1720..1742)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1731..1747
                     /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(1755..1761,1762..1779,1780..1785))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1800..1821
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        1880..2075
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      1926..2061
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2081..2215
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2118..2137)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2172..2191
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2283..2300)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(2454..2919,1..123))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2534..2553)
                     /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 acctgggtcg acattgatta ttgactagta gtggagaatt
     1321 ctgtacaaaa 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 cgaaacacct ctagacccag ctttcttgta caaagttggc attatgcccc
     1681 cgggcgccaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     1741 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     1801 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     1861 ctgtcgtgcc agcggatccg catctcaatt agtcagcaac catagtcccg cccctaactc
     1921 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     1981 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     2041 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct aacttgttta ttgcagctta
     2101 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     2161 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatccgctg
     2221 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     2281 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     2341 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     2401 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     2461 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     2521 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     2581 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     2641 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     2701 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     2761 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     2821 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     2881 ggctacacta gaagaacagt atttggtatc tgcgctctg
//
