LOCUS       sequence-460327-        7074 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A CAGGS driven expression vector containing an nls-mCherry followed 
            by an IRES and then a myristoylated EGFP for labeling the cell 
            membrane. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7074)
  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 7074)
  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-nls-mCherry-stop-IRES-myr-EGFP (JDW 1326)
FEATURES             Location/Qualifiers
     source          1..7074
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(24..41)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_feature    join(53..627,628..630,631..639)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(220..237)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     447..466
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(700..702,703..705,706..1419)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(745..766)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1006..1025)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1353..1374
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1505..1524)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1570..1625
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1624..1643)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(1986..2002)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1986..2002)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1999..2021)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2010..2026
                     /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(2034..2040,2041..2058,2059..2064))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2079..2100
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2159..2354
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2205..2340
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2360..2494
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2397..2416)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2451..2470
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2562..2579)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2733..3321)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2813..2832)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3492..4283,4284..4352))
                     /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     4115..4134
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4353..4457)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        4488..4867
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          5212..6228
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     6152..6174
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     6236..6255
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    6296..6320
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             6332..6352
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6353..7063
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGREFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDIPSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(6498..6516)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6766..6785
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(join(7065..7074,1..15))
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 cttgtacaaa gtggttcgat atcctatagt gtcacctaaa taattccgcc cccccctctc
       61 cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg
      121 tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg
      181 gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag
      241 gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt
      301 ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc
      361 aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga
      421 gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga
      481 aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct
      541 ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg
      601 ttttcctttg aaaaacacga tgataatatg gccacaacca tgctgtgctg tatgagaaga
      661 accaaacagg ttgaaaagaa tgatgaggac caaaagatca tggtgagcaa gggcgaggag
      721 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
      781 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
      841 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
      901 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
      961 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     1021 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     1081 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     1141 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggc gaacttcaag
     1201 atccgccaca acatcgagga cggcggcgtg cagctcgccg accactacca gcagaacacc
     1261 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     1321 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     1381 gccgggatca ctctcggcat ggacgagctg tacaagtaag cggccgcact cctcaggtgc
     1441 aggctgccta tcagaaggtg gtggctggtg tggccaatgc cctggctcac aaataccact
     1501 gagatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt gagcatctga
     1561 cttctggcta ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc
     1621 tctcactcgg aaggacatat gggagggcaa atcatttaaa acatcagaat gagtatttgg
     1681 tttagagttt ggcaacatat gcccatatgc tggctgccat gaacaaaggt tggctataaa
     1741 gaggtcatca gtatatgaaa cagccccctg ctgtccattc cttattccat agaaaagcct
     1801 tgacttgagg ttagattttt tttatatttt gttttgtgtt atttttttct ttaacatccc
     1861 taaaattttc cttacatgtt ttactagcca gatttttcct cctctcctga ctactcccag
     1921 tcatagctgt ccctcttctc ttatggagat ccctcgacct gcagcccaag cttggcgtaa
     1981 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     2041 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     2101 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc
     2161 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     2221 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     2281 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     2341 taggcttttg caaaaagcta acttgtttat tgcagcttat aatggttaca aataaagcaa
     2401 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     2461 caaactcatc aatgtatctt atcatgtctg gatccgctgc attaatgaat cggccaacgc
     2521 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     2581 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     2641 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     2701 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     2761 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     2821 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     2881 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     2941 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     3001 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     3061 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     3121 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     3181 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     3241 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     3301 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     3361 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     3421 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     3481 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     3541 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     3601 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     3661 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     3721 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     3781 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     3841 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     3901 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     3961 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     4021 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     4081 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     4141 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     4201 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     4261 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     4321 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     4381 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     4441 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgggtcgac attgattatt
     4501 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
     4561 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
     4621 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
     4681 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     4741 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     4801 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     4861 taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
     4921 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
     4981 gggggggggg gggggccagg gggggggggg gggggggggg gggggggggg gggggggggg
     5041 gggggggggg gggggcgcgc gcgggggggg gcggggcggg gcggggggcg gggcggggcg
     5101 aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg
     5161 gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct
     5221 gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc
     5281 tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt
     5341 aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag
     5401 gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg
     5461 tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc
     5521 ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc
     5581 ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg
     5641 gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc
     5701 cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg
     5761 ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct
     5821 cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga
     5881 ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt
     5941 cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc
     6001 gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt
     6061 gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac
     6121 ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc
     6181 tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac
     6241 gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcctcgagat atcaaacaag
     6301 tttgtacaaa aaagcaggct acaccatggc tccaaagaag aagcgtaagg taatggtgag
     6361 caagggcgag gaggacaaca tggccatcat caaggagttc atgcgcttca aggtgcacat
     6421 ggagggctcc gtgaacggcc gcgagttcga gatcgagggc gagggcgagg gccgccccta
     6481 cgagggcacc cagaccgcca agctgaaggt gaccaagggc ggccccctgc ccttcgcctg
     6541 ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga
     6601 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     6661 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt
     6721 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa
     6781 gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa
     6841 gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg ccgaggtcaa
     6901 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa
     6961 gctggacatc ccctcccaca acgaggacta caccatcgtg gaacagtacg agcgcgccga
     7021 gggccgccac tccaccggcg gcatggacga gctgtacaag taagacccag cttt
//
