LOCUS       sequence-190869-        4189 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mCardinal from the EF1a promoter in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4189)
  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 4189)
  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-mCardinal
FEATURES             Location/Qualifiers
     source          1..4189
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(358..1152)
                     /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(358..1152)
                     /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(469..488)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1079..1098
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     polyA_signal    1337..1561
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1550..1567
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     1900..1919
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    1918..1973
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     2019..2038
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(2115..2849)
                     /codon_start=1
                     /product="far-red fluorescent protein derived from 
                     mNeptune, with further red-shifted spectra"
                     /label=mCardinal
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTTEGEGKPYEGTQTQ
                     RIKVVEGGPLPFAFDILATCFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTVTQDTSLQDGCLIYNVKLRGVNFPSNGPVMQKKTLGWEATTETLYPADGGLEGRCD
                     MALKLVGGGHLHCNLKTTYRSKKPAKNLKMPGVYFVDRRLERIKEADNETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     regulatory      2846..2855
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     LTR             2893..3161
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     promoter        complement(3174..3385)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     protein_bind    complement(3537..3570)
                     /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(join(3634..4189,1..33))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3714..3733)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
       61 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
      121 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttagcacgtg ctattattga
      181 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
      241 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgatgc ggtgtgaaat
      301 accgcacaga tgcgtaagga gaaaataccg catcaggaaa ttgtaagcgt taataattca
      361 gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc
      421 gtaaagcacg aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt
      481 agccaacgct atgtcctgat agcggtccgc cacccccagc cggccacagt cgatgaatcc
      541 agaaaagcgg ccattttcca ccatgatatt cggcaagcag gcatcgccat gggtcacgac
      601 gagatcctcg ccgtcgggca tgctcgcctt gagcctggcg aacagttcgg ctggcgcgag
      661 cccctgatgc tcttcgtcca gatcatcctg atcgacaaga ccggcttcca tccgagtacg
      721 tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt
      781 atgcagccgc cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtgaga
      841 tgacaggaga tcctgccccg gcacttcgcc caatagcagc cagtcccttc ccgcttcagt
      901 gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc
      961 tgcctcgtct tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg
     1021 gcgcccctgc gctgacagcc ggaacacggc ggcatcagag cagccgattg tctgttgtgc
     1081 ccagtcatag ccgaatagcc tctccaccca agcggccgga gaacctgcgt gcaatccatc
     1141 ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga tcagagcttg atcccctgcg
     1201 ccatcagatc cttggcggca agaaagccat ccagtttact ttgcagggct tcccaacctt
     1261 accagagggc gccccagctg gcaattccgg ttcgcttgct gtccataaaa ccgcccagta
     1321 gaagaggcgc gccaaaccat agagcccacc gcatccccag catgcctgct attgtcttcc
     1381 caatcctccc ccttgctgtc ctgccccacc ccacccccca gaatagaatg acacctactc
     1441 agacaatgcg atgcaatttc ctcattttat taggaaagga cagtgggagt ggcaccttcc
     1501 agggtcaagg aaggcacggg ggaggggcaa acaacagatg gctggcaact agaaggcaca
     1561 gtcgagggct aggggaccct cagttagcct cccccatctc ccgagaagag ggacagctat
     1621 gactgggagt agtcaggaga ggaggaaaaa tctggctagt aaaacatgta aggaaaattt
     1681 tagggatgtt aaagaaaaaa ataacacaaa acaaaatata aaaaaaatct aacctcaagt
     1741 caaggctttt ctatggaata aggaatggac agcagggggc tgtttcatat actgatgacc
     1801 tctttatagc caacctttgt tcatggcagc cagcatatgg gcatatgttg ccaaactcta
     1861 aaccaaatac tcattctgat gttttaaatg atttgccctc ccatatgtcc ttccgagtga
     1921 gagacacaaa aaattccaac acactattgc aatgaaaata aatttccttt attagccaga
     1981 agtcagatgc tcaaggggct tcatgatgtc cccataattt ttggcagagg gaaaaagatc
     2041 tcagtggtat ttgtgagcca gggtattggc cacaccagcc accaccttct gataggcagc
     2101 ctgcacgcgg ccgcttactt gtacagctcg tccatgccat taagtttgtg ccccagtttg
     2161 ctagggaggt cgcagtatct ggccacagcc acctcgtgct gctcgacgta ggtctcattg
     2221 tcggcctcct tgattctttc cagtctgcgg tccacaaagt agacgccggg catcttgagg
     2281 ttcttagcgg gtttcttgga tctgtatgtg gtcttcaggt tgcagtgcag gtggcccccg
     2341 cccacgagct tcagggccat gtcgcatctg ccttccaggc cgccgtcagc ggggtacagg
     2401 gtctcggtgg tggcctccca gccgagtgtt ttcttctgca tcacagggcc gttggatggg
     2461 aagttcaccc ctctgagctt gacgttgtag atcaagcagc cgtcctggag gctggtgtcc
     2521 tgggtaacgg taagcacgcc cccgtcttcg tatgtggtga ctctctccca tgtgaagccc
     2581 tcagggaagg actgcttaaa gaaatcgggg atgccctggg tgtggttgat gaaggtcttg
     2641 ctcccgtaca taaagcaggt ggccaggatg tcgaatgcga acggcagggg gcctccctcc
     2701 accaccttaa tcctctgggt ctgggtgccc tcgtagggct tgccctcccc ttcggtggtg
     2761 cacttgaagt ggtggttgtt cacggtgcct tccatgtaca gcttcatgtg catgttctcc
     2821 ttgatcagct cctcgccctt gctcaccatg gtggcgaccg gtagcgctag cgtaggcgcc
     2881 ggtcacagct tggatctgta acggcgcaga acagaaaacg aaacaaagac gtagagttga
     2941 gcaagcaggg tcaggcaaag cgtggagagc cggctgagtc taggtaggct ccaagggagc
     3001 gccggacaaa ggcccggtct cgacctgagc tttaaactta cctagacggc ggacgcagtt
     3061 caggaggcac cacaggcggg aggcggcaga acgcgactca accggcgtgg atggcggcct
     3121 caggtagggc ggcgggcgcg tgaaggagag atgcgagccc ctcgaagctt cagctgtgtt
     3181 ctggcggcaa acccgttgcg aaaaagaacg ttcacggcga ctactgcact tatatacggt
     3241 tctcccccac cctcgggaaa aaggcggagc cagtacacga catcactttc ccagtttacc
     3301 ccgcgccacc ttctctaggc accggttcaa ttgccgaccc ctccccccaa cttctcgggg
     3361 actgtgggcg atgtgcgctc tgcccactga cgggcaccgg agcgatcgca gatcgtcaat
     3421 gggcgggggt cgttgggcgg tcagccaggc gggccattta ccgtaagtta tgtaacgcgg
     3481 aactccatat atgggctatg aactaatgac cccgtaattg attactatta cttaagataa
     3541 cttcgtatag catacattat acgaagttat gaacatgtga gcaaaaggcc agcaaaaggc
     3601 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3661 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3721 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     3781 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     3841 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     3901 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     3961 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4021 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     4081 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4141 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaag
//
