LOCUS       sequence-247034-        4061 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An entry vector with U6a and U6c promoter driving opa1 guide RNAs 
            expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4061)
  AUTHORS   Zhou W, Hsu AY, Wang Y, Syahirah R, Wang T, Jeffries J, Wang X, 
            Mohammad H, Seleem MN, Umulis D, Deng Q
  TITLE     Mitofusin 2 regulates neutrophil adhesive migration and the actin 
            cytoskeleton.
  JOURNAL   J Cell Sci. 2020 Sep 4;133(17). pii: jcs.248880. doi: 
            10.1242/jcs.248880.
  PUBMED    32788232
REFERENCE   2  (bases 1 to 4061)
  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: "J Cell 
            Sci."; date: "2020-09-4"; volume: "133(17). pii: jcs.248880. doi"; 
            pages: " 10.1242/jcs.248880"
COMMENT     Sequence Label: p3E-U6a-U6c-opa1 guide
FEATURES             Location/Qualifiers
     source          1..4061
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(624..747)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(761..777)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    793..927
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(817..836)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     871..890
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(2170..2265)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2302..2321)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2303..2321)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2326..2342)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2326..2342)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2444..2463)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2455..3264
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2522..2541)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3411..3999
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3900..3919
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggccctg cagctctaga gctcgaattc tacaggtcac
      601 taataccatc taagtagttg gttcatagtg actgcatatg ttgtgtttta cagtattatg
      661 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt
      721 ctcgttcaac tttcttgtac aaagttgaga tctataattc actggccgtc gttttacggt
      781 accatcgatg atgatccaga catgataaga tacattgatg agtttggaca aaccacaact
      841 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
      901 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
      961 gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtatg
     1021 gctgattatg atcctctaga tcccttgggt agcagcgaag tcgaggcatt tctgtcctgg
     1081 ctggtctaga ggtctctcag gtttaaaaaa agcaccgact cggtgccact ttttcaagtt
     1141 gataacggac tagccttatt taaacttgct atgctgtttc cagcatagct cttaaaccct
     1201 cactgctcag ctgcccgaac taggagcctg gagaactgct atataaaccc gggacttctg
     1261 ggtatgtttc tgccatgtgg taagtggata aacctactta ttcaactcat ttacctcaaa
     1321 catgacagta aacctcttcg gggcaaacct gcaaacgctt aaaaccttca aagtttgaaa
     1381 ccagcagttt tgtagaggtt taccgtagta tttttgaact gtaaagcata tgcagaggaa
     1441 gctggtttta ggcttgtgtg attggatgag ttcttctgtg acgtaactga tcttttgtcc
     1501 tcgtgtcctt cgccttcccc cacaggcatg cgcggacctt cccccgccgc agaccagagg
     1561 acaacatgct cagacgccag caaacggccc atcgaaacat tcacagactc tcggattgga
     1621 gagaccgggc ccgaggtctc tgactaaaaa agcaccgact cggtgccact ttttcaagtt
     1681 gataacggac tagccttatt taaacttgct atgctgtttc cagcatagct cttaaaccac
     1741 tctggtcccc aactaccgaa ccaagagctg gagggagagc tatatatacc agggacttct
     1801 gggtatgttt ttgggaggtg gtgagtgact aaaccactta ttcagctccc ttagatcaag
     1861 tctgacccta tatcatggtg acataaacct gcaaactgat aaaacctgaa ggatctcaaa
     1921 tccagagttt gtgtgaggga ttaccgtggt aattttcaaa ctgtaaagca tatgcaaaat
     1981 tatctggtct tggcttgagt gattgggtgt ctcggtgtga tgcagggacg ttttcagtga
     2041 cgtgtccttc tccctccccc acaggcatgc gcagaacatt tcccccctcc ttgaagacca
     2101 gaacaaaaga cgccgagagc aggaaactcg tcttactgaa tgaccgaggc tggagaaatc
     2161 gaccctaggc aactttatta tacaaagttg gcattataaa aaagcattgc ttatcaattt
     2221 gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttggagc tccatggtag
     2281 cgttaacgcg gccgcgatat cccctatagt gagtcgtatt acatggtcat agctgtttcc
     2341 tggcagctct ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata
     2401 tcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc
     2461 catattcaac gggaaacgtc gaggccgcga ttaaattcca acatggatgc tgatttatat
     2521 gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgcttgtat
     2581 gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat
     2641 gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc
     2701 aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggaaaa
     2761 acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg
     2821 gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat
     2881 cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt
     2941 gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa
     3001 cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt
     3061 atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac
     3121 cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag
     3181 aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat
     3241 ttgatgctcg atgagttttt ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc
     3301 attacgctga cttgacggga cggcgcaagc tcatgaccaa aatcccttaa cgtgagttac
     3361 gcgtcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3421 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3481 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3541 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     3601 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     3661 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     3721 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3781 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     3841 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3901 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     3961 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4021 ttacggttcc tggccttttg ctggcctttt gctcacatgt t
//
