LOCUS       sequence-447098-        5402 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway compatible middle entry clone containing a Luciferase P2A 
            H2A-mCherry dual reporter (Luciferase/nuclear red fluorescent 
            reporter).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5402)
  AUTHORS   Gillespie WB 3rd, Zhang Y, Ruiz OE, Cerda J 3rd, Ortiz-Guzman J, 
            Sherman M, Turner WD, Largoza G, Mosser LE, Fujimoto E, Chien CB, 
            Kwan KM, Arenkiel BR, Devine WP, Wythe JD
  TITLE     MultiSite Assembly of Gateway Induced Clones (MAGIC): a flexible 
            cloning toolbox for use in vertebrate model systems.
  JOURNAL   Development. 2025 Nov 15;152(22):dev204308. doi: 10.1242/dev.204308.
            Epub 2025 Nov 13.
  PUBMED    41230856
REFERENCE   2  (bases 1 to 5402)
  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: 
            "Development."; date: "2025-11-15"; volume: "152(22):dev204308. 
            doi"; pages: " 10.1242/dev.204308. Epub 2025 Nov 13"
COMMENT     Sequence Label: pME-Luciferase-P2A-H2A-mCherry (JDW 926)
FEATURES             Location/Qualifiers
     source          1..5402
                     /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     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      681..690
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             687..2336
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     primer_bind     complement(753..771)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     CDS             2346..2402
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2799..3509
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2944..2962)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3212..3231
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3406..3429
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    complement(3527..3626)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3643..3662)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3644..3662)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3667..3683)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3667..3683)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3785..3804)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3796..4605
                     /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(3863..3882)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4752..5340
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5241..5260
                     /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 tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctgg atccactagt gccaccatgg aagacgccaa aaacataaag aaaggcccgg
      721 cgccattcta tccgctggaa gatggaaccg ctggagagca actgcataag gctatgaaga
      781 gatacgccct ggttcctgga acaattgctt ttacagatgc acatatcgag gtggacatca
      841 cttacgctga gtacttcgaa atgtccgttc ggttggcaga agctatgaaa cgatatgggc
      901 tgaatacaaa tcacagaatc gtcgtatgca gtgaaaactc tcttcaattc tttatgccgg
      961 tgttgggcgc gttatttatc ggagttgcag ttgcgcccgc gaacgacatt tataatgaac
     1021 gtgaattgct caacagtatg ggcatttcgc agcctaccgt ggtgttcgtt tccaaaaagg
     1081 ggttgcaaaa aattttgaac gtgcaaaaaa agctcccaat catccaaaaa attattatca
     1141 tggattctaa aacggattac cagggatttc agtcgatgta cacgttcgtc acatctcatc
     1201 tacctcccgg ttttaatgaa tacgattttg tgccagagtc cttcgatagg gacaagacaa
     1261 ttgcactgat catgaactcc tctggatcta ctggtctgcc taaaggtgtc gctctgcctc
     1321 atagaactgc ctgcgtgaga ttctcgcatg ccagagatcc tatttttggc aatcaaatca
     1381 ttccggatac tgcgatttta agtgttgttc cattccatca cggttttgga atgtttacta
     1441 cactcggata tttgatatgt ggatttcgag tcgtcttaat gtatagattt gaagaagagc
     1501 tgtttctgag gagccttcag gattacaaga ttcaaagtgc gctgctggtg ccaaccctat
     1561 tctccttctt cgccaaaagc actctgattg acaaatacga tttatctaat ttacacgaaa
     1621 ttgcttctgg tggcgctccc ctctctaagg aagtcgggga agcggttgcc aagaggttcc
     1681 atctgccagg tatcaggcaa ggatatgggc tcactgagac tacatcagct attctgatta
     1741 cacccgaggg ggatgataaa ccgggcgcgg tcggtaaagt tgttccattt tttgaagcga
     1801 aggttgtgga tctggatacc gggaaaacgc tgggcgttaa tcaaagaggc gaactgtgtg
     1861 tgagaggtcc tatgattatg tccggttatg taaacaatcc ggaagcgacc aacgccttga
     1921 ttgacaagga tggatggcta cattctggag acatagctta ctgggacgaa gacgaacact
     1981 tcttcatcgt tgaccgcctg aagtctctga ttaagtacaa aggctatcag gtggctcccg
     2041 ctgaattgga atccatcttg ctccaacacc ccaacatctt cgacgcaggt gtcgcaggtc
     2101 ttcccgacga tgacgccggt gaacttcccg ccgccgttgt tgttttggag cacggaaaga
     2161 cgatgacgga aaaagagatc gtggattacg tcgccagtca agtaacaacc gcgaaaaagt
     2221 tgcgcggagg agttgtgttt gtggacgaag taccgaaagg tcttaccgga aaactcgacg
     2281 caagaaaaat cagagagatc ctcataaagg ccaagaaggg cggaaagatc gccgtgggta
     2341 ccggagccac taacttctcc ctgttgaaac aagcagggga tgtcgaagag aatcccgggc
     2401 cacctaggat ggcaggtgga aaagcaggta aagacagtgg aaaagccaag acgaaagccg
     2461 tctccagatc tcagagagct ggtctgcagt tccctgtagg tcgtatccac aggcacctga
     2521 agacacgaac caccagccat ggccgtgtcg gagctacagc tgcggtttac agtgctgcta
     2581 tcctggagta tctcaccgct gaggtgctgg agttggctgg aaacgcttct aaggatctga
     2641 aggtgaagcg aatcactccc agacatttac agctggccat ccgcggagac gaggagctgg
     2701 actcgcttat caaggccacc attgctggag gaggagtcat tcctcacatc cacaagtctc
     2761 tgattggtaa gaagggccag cagaaaaccg caggggggat ggtgagcaag ggcgaggagg
     2821 acaacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga
     2881 acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga
     2941 ccgccaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc
     3001 ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact
     3061 tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg
     3121 gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga
     3181 agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct
     3241 gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc
     3301 agaggctgaa gctgaaggac ggcggccact acgacgccga ggtcaagacc acctacaagg
     3361 ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagctg gacatcacct
     3421 cccacaacga ggactacacc atcgtggaac agtacgagcg cgccgagggc cgccactcca
     3481 ccggcggcat ggacgagctg tacaagtaat gatagctcga gagatctccc agctttcttg
     3541 tacaaagttg gcattataag aaagcattgc ttatcaattt gttgcaacga acaggtcact
     3601 atcagtcaaa ataaaatcat tatttgccat ccagctgata tcccctatag tgagtcgtat
     3661 tacatggtca tagctgtttc ctggcagctc tggcccgtgt ctcaaaatct ctgatgttac
     3721 attgcacaag ataaaaatat atcatcatga acaataaaac tgtctgctta cataaacagt
     3781 aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cgaggccgcg attaaattcc
     3841 aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt
     3901 gcgacaatct atcgcttgta tgggaagccc gatgcgccag agttgtttct gaaacatggc
     3961 aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa
     4021 tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc
     4081 accactgcga tccccggaaa aacagcattc caggtattag aagaatatcc tgattcaggt
     4141 gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt
     4201 aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat
     4261 aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa
     4321 gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt cactcatggt
     4381 gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt
     4441 ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt
     4501 gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat
     4561 atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcaga attggttaat
     4621 tggttgtaac actggcagag cattacgctg acttgacggg acggcgcaag ctcatgacca
     4681 aaatccctta acgtgagtta cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc
     4741 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4801 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     4861 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     4921 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     4981 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     5041 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     5101 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     5161 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     5221 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     5281 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     5341 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     5401 tt
//
