LOCUS       sequence-460483-        7722 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A CAGGS driven luciferase reporter followed by a P2A cleavage 
            peptide and an H2A mCherry cassette for nuclear labeling..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7722)
  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 7722)
  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: pCAGEN-Luciferase-P2A-H2A-mCherry (JDW 1129)
FEATURES             Location/Qualifiers
     source          1..7722
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          661..1677
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1601..1623
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1685..1704
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1744..1768
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1781..1790
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1787..3436
                     /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(1853..1871)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     CDS             3446..3502
                     /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             3899..4609
                     /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(4044..4062)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4312..4331
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4506..4529
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    complement(4627..4651)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(4743..4762)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4808..4863
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4862..4881)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5224..5240)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5224..5240)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5237..5259)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5248..5264
                     /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(5272..5278,5279..5296,5297..5302))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5317..5338
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5397..5592
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5443..5578
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5598..5732
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5635..5654)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5689..5708
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5800..5817)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5971..6559)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6051..6070)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6730..7521,7522..7590))
                     /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     7353..7372
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7591..7695)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      421 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      481 cgatgggggc gggggggggg ggggggcgcg ccrggggggg gggggggggg gggggggggg
      541 gggggggsgr ggsggrgagg kgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
      601 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
      661 ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg
      721 ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct
      781 ccgggctgta attagcgctt ggtttaatga cggcttgttt cttttctgtg gctgcgtgaa
      841 agccttgagg ggctccggga gggccctttg tgcgggggga gcggctcggg gggtgcgtgc
      901 gtgtgtgtgt gcgtggggag cgccgcgtgc ggctccgcgc tgcccggcgg ctgtgagcgc
      961 tgcgggcgcg gcgcggggct ttgtgcgctc cgcagtgtgc gcgaggggag cgcggccggg
     1021 ggcggtgccc cgcggtgcgg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg
     1081 tgcgtggggg ggtgagcagg gggtgtgggc gcgtcggtcg ggctgcaacc ccccctgcac
     1141 ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg tacggggcgt
     1201 ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg kgggggtgcc gggcggggcg
     1261 gggccgcckc gggccggggr gggctcgggg grggggcgcg gcggcccccg gagcgccggc
     1321 ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg
     1381 cagggacttc ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc
     1441 ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag
     1501 ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg gggctgtccg
     1561 cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg
     1621 accggcggct ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc
     1681 ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaagaatt cctcgaggat
     1741 atcacaagtt tgtacaaaaa agcaggctgg atccactagt gccaccatgg aagacgccaa
     1801 aaacataaag aaaggcccgg cgccattcta tccgctggaa gatggaaccg ctggagagca
     1861 actgcataag gctatgaaga gatacgccct ggttcctgga acaattgctt ttacagatgc
     1921 acatatcgag gtggacatca cttacgctga gtacttcgaa atgtccgttc ggttggcaga
     1981 agctatgaaa cgatatgggc tgaatacaaa tcacagaatc gtcgtatgca gtgaaaactc
     2041 tcttcaattc tttatgccgg tgttgggcgc gttatttatc ggagttgcag ttgcgcccgc
     2101 gaacgacatt tataatgaac gtgaattgct caacagtatg ggcatttcgc agcctaccgt
     2161 ggtgttcgtt tccaaaaagg ggttgcaaaa aattttgaac gtgcaaaaaa agctcccaat
     2221 catccaaaaa attattatca tggattctaa aacggattac cagggatttc agtcgatgta
     2281 cacgttcgtc acatctcatc tacctcccgg ttttaatgaa tacgattttg tgccagagtc
     2341 cttcgatagg gacaagacaa ttgcactgat catgaactcc tctggatcta ctggtctgcc
     2401 taaaggtgtc gctctgcctc atagaactgc ctgcgtgaga ttctcgcatg ccagagatcc
     2461 tatttttggc aatcaaatca ttccggatac tgcgatttta agtgttgttc cattccatca
     2521 cggttttgga atgtttacta cactcggata tttgatatgt ggatttcgag tcgtcttaat
     2581 gtatagattt gaagaagagc tgtttctgag gagccttcag gattacaaga ttcaaagtgc
     2641 gctgctggtg ccaaccctat tctccttctt cgccaaaagc actctgattg acaaatacga
     2701 tttatctaat ttacacgaaa ttgcttctgg tggcgctccc ctctctaagg aagtcgggga
     2761 agcggttgcc aagaggttcc atctgccagg tatcaggcaa ggatatgggc tcactgagac
     2821 tacatcagct attctgatta cacccgaggg ggatgataaa ccgggcgcgg tcggtaaagt
     2881 tgttccattt tttgaagcga aggttgtgga tctggatacc gggaaaacgc tgggcgttaa
     2941 tcaaagaggc gaactgtgtg tgagaggtcc tatgattatg tccggttatg taaacaatcc
     3001 ggaagcgacc aacgccttga ttgacaagga tggatggcta cattctggag acatagctta
     3061 ctgggacgaa gacgaacact tcttcatcgt tgaccgcctg aagtctctga ttaagtacaa
     3121 aggctatcag gtggctcccg ctgaattgga atccatcttg ctccaacacc ccaacatctt
     3181 cgacgcaggt gtcgcaggtc ttcccgacga tgacgccggt gaacttcccg ccgccgttgt
     3241 tgttttggag cacggaaaga cgatgacgga aaaagagatc gtggattacg tcgccagtca
     3301 agtaacaacc gcgaaaaagt tgcgcggagg agttgtgttt gtggacgaag taccgaaagg
     3361 tcttaccgga aaactcgacg caagaaaaat cagagagatc ctcataaagg ccaagaaggg
     3421 cggaaagatc gccgtgggta ccggagccac taacttctcc ctgttgaaac aagcagggga
     3481 tgtcgaagag aatcccgggc cacctaggat ggcaggtgga aaagcaggta aagacagtgg
     3541 aaaagccaag acgaaagccg tctccagatc tcagagagct ggtctgcagt tccctgtagg
     3601 tcgtatccac aggcacctga agacacgaac caccagccat ggccgtgtcg gagctacagc
     3661 tgcggtttac agtgctgcta tcctggagta tctcaccgct gaggtgctgg agttggctgg
     3721 aaacgcttct aaggatctga aggtgaagcg aatcactccc agacatttac agctggccat
     3781 ccgcggagac gaggagctgg actcgcttat caaggccacc attgctggag gaggagtcat
     3841 tcctcacatc cacaagtctc tgattggtaa gaagggccag cagaaaaccg caggggggat
     3901 ggtgagcaag ggcgaggagg acaacatggc catcatcaag gagttcatgc gcttcaaggt
     3961 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
     4021 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggcggcc ccctgccctt
     4081 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc
     4141 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt
     4201 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
     4261 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
     4321 gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc
     4381 cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgccga
     4441 ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa
     4501 catcaagctg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgagcg
     4561 cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtaat gatagctcga
     4621 gagatctccc agctttcttg tacaaagtgg tgatatcgcg gccgcactcc tcaggtgcag
     4681 gctgcctatc agaaggtggt ggctggtgtg gccaatgccc tggctcacaa ataccactga
     4741 gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact
     4801 tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc
     4861 tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt
     4921 tagagtttgg caacatatgc ccatatgctg gctgccatga acaaaggttg gctataaaga
     4981 ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag aaaagccttg
     5041 acttgaggtt agattttttt tatattttgt tttgtgttat ttttttcttt aacatcccta
     5101 aaattttcct tacatgtttt actagccaga tttttcctcc tctcctgact actcccagtc
     5161 atagctgtcc ctcttctctt atggagatcc ctcgacctgc agcccaagct tggcgtaatc
     5221 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     5281 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     5341 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc ggatccgcat
     5401 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     5461 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     5521 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     5581 ggcttttgca aaaagctaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     5641 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     5701 aactcatcaa tgtatcttat catgtctgga tccgctgcat taatgaatcg gccaacgcgc
     5761 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5821 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5881 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5941 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     6001 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     6061 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     6121 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     6181 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6241 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6301 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6361 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6421 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6481 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6541 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6601 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6661 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6721 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6781 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6841 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6901 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6961 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     7021 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     7081 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     7141 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     7201 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     7261 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     7321 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     7381 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7441 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7501 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7561 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7621 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7681 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gg
//
