LOCUS       sequence-192219-        4111 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mKOk from the EF1a promoter in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4111)
  AUTHORS   Geisinger JM, Turan S, Hernandez S, Spector LP, Calos MP
  TITLE     In vivo blunt-end cloning through CRISPR/Cas9-facilitated 
            non-homologous end-joining.
  JOURNAL   Nucleic Acids Res. 2016 Jan 13. pii: gkv1542.
  PUBMED    26762978
REFERENCE   2  (bases 1 to 4111)
  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: "Nucleic 
            Acids Res. 2016 Jan 13. pii: gkv1542."
COMMENT     Sequence Label: pKER-mKOk
FEATURES             Location/Qualifiers
     source          1..4111
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    164..388
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     377..394
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     727..746
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    745..800
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     846..865
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(942..1598)
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQFKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     regulatory      1595..1604
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     LTR             1642..1910
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     promoter        complement(1923..2134)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     protein_bind    complement(2286..2319)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(2383..2971)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2463..2482)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3296..4090)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(3296..4090)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3407..3426)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4017..4036
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 ctcttgatca gagcttgatc ccctgcgcca tcagatcctt ggcggcaaga aagccatcca
       61 gtttactttg cagggcttcc caaccttacc agagggcgcc ccagctggca attccggttc
      121 gcttgctgtc cataaaaccg cccagtagaa gaggcgcgcc aaaccataga gcccaccgca
      181 tccccagcat gcctgctatt gtcttcccaa tcctccccct tgctgtcctg ccccacccca
      241 ccccccagaa tagaatgaca cctactcaga caatgcgatg caatttcctc attttattag
      301 gaaaggacag tgggagtggc accttccagg gtcaaggaag gcacggggga ggggcaaaca
      361 acagatggct ggcaactaga aggcacagtc gagggctagg ggaccctcag ttagcctccc
      421 ccatctcccg agaagaggga cagctatgac tgggagtagt caggagagga ggaaaaatct
      481 ggctagtaaa acatgtaagg aaaattttag ggatgttaaa gaaaaaaata acacaaaaca
      541 aaatataaaa aaaatctaac ctcaagtcaa ggcttttcta tggaataagg aatggacagc
      601 agggggctgt ttcatatact gatgacctct ttatagccaa cctttgttca tggcagccag
      661 catatgggca tatgttgcca aactctaaac caaatactca ttctgatgtt ttaaatgatt
      721 tgccctccca tatgtccttc cgagtgagag acacaaaaaa ttccaacaca ctattgcaat
      781 gaaaataaat ttcctttatt agccagaagt cagatgctca aggggcttca tgatgtcccc
      841 ataatttttg gcagagggaa aaagatctca gtggtatttg tgagccaggg tattggccac
      901 accagccacc accttctgat aggcagcctg cacgcggccg cctaggaatg agctactgca
      961 tcttctactt gctcagtaat gttgccttcg gttttcctga cgaggcgatg accgatgtaa
     1021 tgatctcctg gcatttcaag aatttctttt gccgccttgt aagtagtctt gaattggcat
     1081 ttgtgattgc cgcctccttc aagttttagg tacatcgtaa catcaccctt cagaactccg
     1141 tcgctggcag taattttctc ggttgatggc tcccaatcaa cactttggtt ttgcatgata
     1201 ggaccatcgg caggaaagtt aaccccagta aatttggatt tgtggtagaa ggtgtttcct
     1261 ctaaggctta tatgcgcact gactgaagcg gacccaccat cttcgaactc caacgacctt
     1321 tcccatgaca ggccttcagg aaatgcttgt ttgaaatagt ctggtatctc ttctggatat
     1381 ttagtaaaaa ctctgtggcc gtaacagaac acgtgtgaca ctaagtcaaa cgcgaaaggc
     1441 attggcccgc cttcggccat tgtgacgcgt agtgtcatct cttgatgtcc ctcgtaaggt
     1501 ctgcctgtgc cttcaccttc aattgtgaac tcatgcccat tgacggagcc gtccatgtag
     1561 tacctcatct tcatctctgg tttaatcaca ctcaccatgg tggcgaccgg tagcgctagc
     1621 gtaggcgccg gtcacagctt ggatctgtaa cggcgcagaa cagaaaacga aacaaagacg
     1681 tagagttgag caagcagggt caggcaaagc gtggagagcc ggctgagtct aggtaggctc
     1741 caagggagcg ccggacaaag gcccggtctc gacctgagct ttaaacttac ctagacggcg
     1801 gacgcagttc aggaggcacc acaggcggga ggcggcagaa cgcgactcaa ccggcgtgga
     1861 tggcggcctc aggtagggcg gcgggcgcgt gaaggagaga tgcgagcccc tcgaagcttc
     1921 agctgtgttc tggcggcaaa cccgttgcga aaaagaacgt tcacggcgac tactgcactt
     1981 atatacggtt ctcccccacc ctcgggaaaa aggcggagcc agtacacgac atcactttcc
     2041 cagtttaccc cgcgccacct tctctaggca ccggttcaat tgccgacccc tccccccaac
     2101 ttctcgggga ctgtgggcga tgtgcgctct gcccactgac gggcaccgga gcgatcgcag
     2161 atcgtcaatg ggcgggggtc gttgggcggt cagccaggcg ggccatttac cgtaagttat
     2221 gtaacgcgga actccatata tgggctatga actaatgacc ccgtaattga ttactattac
     2281 ttaagataac ttcgtatagc atacattata cgaagttatg aacatgtgag caaaaggcca
     2341 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     2401 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     2461 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     2521 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     2581 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     2641 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     2701 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     2761 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     2821 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     2881 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     2941 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     3001 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     3061 gatcttcacc tagatccttt taaattaaaa atgaagtttt agcacgtgct attattgaag
     3121 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     3181 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgatgcgg tgtgaaatac
     3241 cgcacagatg cgtaaggaga aaataccgca tcaggaaatt gtaagcgtta ataattcaga
     3301 agaactcgtc aagaaggcga tagaaggcga tgcgctgcga atcgggagcg gcgataccgt
     3361 aaagcacgag gaagcggtca gcccattcgc cgccaagctc ttcagcaata tcacgggtag
     3421 ccaacgctat gtcctgatag cggtccgcca cccccagccg gccacagtcg atgaatccag
     3481 aaaagcggcc attttccacc atgatattcg gcaagcaggc atcgccatgg gtcacgacga
     3541 gatcctcgcc gtcgggcatg ctcgccttga gcctggcgaa cagttcggct ggcgcgagcc
     3601 cctgatgctc ttcgtccaga tcatcctgat cgacaagacc ggcttccatc cgagtacgtg
     3661 ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca ggtagccgga tcaagcgtat
     3721 gcagccgccg cattgcatca gccatgatgg atactttctc ggcaggagca aggtgagatg
     3781 acaggagatc ctgccccggc acttcgccca atagcagcca gtcccttccc gcttcagtga
     3841 caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc cagccacgat agccgcgctg
     3901 cctcgtcttg cagttcattc agggcaccgg acaggtcggt cttgacaaaa agaaccgggc
     3961 gcccctgcgc tgacagccgg aacacggcgg catcagagca gccgattgtc tgttgtgccc
     4021 agtcatagcc gaatagcctc tccacccaag cggccggaga acctgcgtgc aatccatctt
     4081 gttcaatcat gcgaaacgat cctcatcctg t
//
