LOCUS       pCAG-MitoRlucN-T2A-RlucCER-IRES-mCherry        7428 bp DNA     circular SYN 23-APR-2024
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7428)
  AUTHORS   Ginam Cho
  TITLE     Direct Submission
  JOURNAL   Exported Apr 23, 2024 from SnapGene Viewer 7.2.0
            https://www.snapgene.com
COMMENT     Sequence Label: pCAG-MitoRlucN-T2A-RlucCER-IRES-mCherry
COMMENT     Alias: pCAG_split-Rluc_T2A_mCherry
FEATURES             Location/Qualifiers
     source          1..7428
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
                     /note="color: #ffffff"
     primer_bind     4..47
                     /label=CMVa2
                     /note="color: black; sequence: 
                     gacattgattattgactagttattaatagtaatcaattacgggg"
     promoter        386..661
                     /label=chicken beta-actin promoter
                     /note="color: #ffffff; direction: RIGHT"
     intron          662..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
                     /note="color: #------"
     primer_bind     1696..1743
                     /label=mitoF
                     /note="color: black; sequence: 
                     tgctgtctcatcattttggcaaagcaccATGGCCATCCAGCTGCGGTC; added: 
                     2022-11-22"
     CDS             1725..1882
                     /codon_start=3
                     /label=mito-tag
                     /note="color: #ffff00"
                     /translation="AIQLRSLFPLALPGLLALLGWWWFFSRKKDLELKLRILQSTVPRA
                     RDPPVAT"
     CDS             1883..1906
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /note="color: #cc99b2"
                     /translation="DYKDDDDK"
     CDS             1907..2185
                     /label=sLuc
                     /note="color: #99cc00; direction: RIGHT"
     primer_bind     2161..2205
                     /label=T2AF
                     /note="color: black; sequence: 
                     GTCCGGCAAGAGCGGGAATGGCTCAGCTtcgagtggaGAGGGCAG; added: 
                     2022-11-22"
     primer_bind     complement(2161..2185)
                     /label=mitoR
                     /note="color: black; sequence: TGAGCCATTCCCGCTCTTGccggac; 
                     added: 2022-11-22"
     CDS             2198..2251
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /note="color: #cc99b2
                     Cleavage site after base 2248"
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     2227..2275
                     /label=cRlucF
                     /note="color: black; sequence: 
                     TGACGTGGAGGAGAATCCCGGCCCTtatcgcctcctggatcactacaag; added: 
                     2022-11-23"
     primer_bind     complement(2235..2251)
                     /label=T2AR
                     /note="color: black; sequence: AGGGCCGGGATTCTCCT; added: 
                     2022-11-22"
     CDS             2252..2990
                     /codon_start=1
                     /label=cRluc
                     /note="color: #ff99cc"
                     /translation="YRLLDHYKYLTAWFELLNLPKKIIFVGHDWGAALAFHYAYEHQDR
                     IKAIVHMESVVDVIESWDEWPDIEEDIALIKSEEGEKMVLENNFFVETVLPSKIMRKLE
                     PEEFAAYLEPFKEKGEVRRPTLSWPREIPLVKGGKPDVVQIVRNYNAYLRASDDLPKLF
                     IESDPGFFSNAIVEGAKKFPNTEFVKVKGLHFLQEDAPDEMGKYIKSFVERVLKNEQAS
                     EQKLISEEDLSGLRSRAQASNSRV"
     CDS             2918..2947
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /note="color: #cc99b2"
                     /translation="EQKLISEEDL"
     misc_feature    2948..2989
                     /label=linker
                     /note="color: #ffff40"
     CDS             2990..3043
                     /label=ER targeting
                     /note="color: #20ff20; direction: RIGHT"
     primer_bind     complement(3018..3063)
                     /label=ERR
                     /note="color: black; sequence: 
                     cccctagaagcttctgcagatcatttcatgaacaagccaacgaaca; added: 
                     2022-11-22"
     misc_feature    join(3069..3643,3644..3646,3647..3655)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
                     /note="This feature has 3 segments:
                        1: 3069 .. 3643 / #ccffff
                        2: 3644 .. 3646 / #a6acb3 / ATG
                        3: 3647 .. 3655 / #a6acb3"
     primer_bind     complement(3634..3665)
                     /label=IRES_R
                     /note="color: black; sequence: 
                     tgctcaccatggttgtggccatattatcatcg; added: 2022-09-01"
     primer_bind     complement(3634..3655)
                     /label=IRES reverse for PCR cloning
                     /note="color: black; sequence: ggttgtggccatattatcatcg"
     regulatory      3653..3659
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
                     /regulatory_class="other"
     CDS             3656..4366
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /note="color: #ff0000"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3801..3819)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
                     /note="color: black; sequence: TTGGTCACCTTCAGCTTGG"
     primer_bind     4069..4088
                     /label=mCherry-F
                     /note="mCherry, forward primer"
                     /note="color: black; sequence: CCCCGTAATGCAGAAGAAGA"
     primer_bind     complement(4355..4392)
                     /label=mCh_R
                     /note="color: black; sequence: 
                     agcctgcacctgaggagtgcggccgcttacttgtacag; added: 2022-09-01"
     misc_feature    4385..4885
                     /label=rb_glob pA
                     /note="color: #c0c0c0"
     polyA_site      4517..4572
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal"
                     /note="color: #a6acb3"
     rep_origin      complement(5678..6266)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     CDS             complement(join(6437..7228,7229..7297))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 6437 .. 7228 / #ccffcc
                        2: 7229 .. 7297 / #ccffcc / signal sequence
                     Cleavage site after base 7228"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7298..7402)
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: LEFT"
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 tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
     1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     1681 acgtgctggt tattgtgctg tctcatcatt ttggcaaagc accatggcca tccagctgcg
     1741 gtctctcttt ccactggcct tgccaggttt gctggctctc ctgggatggt ggtggttttt
     1801 cagccgcaag aaggacctgg aacttaagct gcggattctg cagtcaaccg tgccccgcgc
     1861 aagagaccct cccgtcgcaa ctgattacaa ggatgacgac gataaggaat tcatggcttc
     1921 caaggtgtac gaccccgagc aacgcaaacg catgatcact gggcctcagt ggtgggctcg
     1981 ctgcaagcaa atgaacgtgc tggactcctt catcaactac tatgattccg agaagcacgc
     2041 cgagaacgcc gtgatttttc tgcatggtaa cgctacctcc agctacctgt ggaggcacgt
     2101 cgtgcctcac atcgagcccg tggctagatg catcatccct gatctgatcg gaatgggtaa
     2161 gtccggcaag agcgggaatg gctcagcttc gagtggagag ggcagaggaa gtcttctaac
     2221 atgcggtgac gtggaggaga atcccggccc ttatcgcctc ctggatcact acaagtacct
     2281 caccgcttgg ttcgagctgc tgaaccttcc aaagaaaatc atctttgtgg gccacgactg
     2341 gggggctgct ctggcctttc actacgccta cgagcaccaa gacaggatca aggccatcgt
     2401 ccatatggag agtgtcgtgg acgtgatcga gtcctgggac gagtggcctg acatcgagga
     2461 ggatatcgcc ctgatcaaga gcgaagaggg cgagaaaatg gtgcttgaga ataacttctt
     2521 cgtcgagacc gtgctcccaa gcaagatcat gcggaaactg gagcctgagg agttcgctgc
     2581 ctacctggag ccattcaagg agaagggcga ggttagacgg cctaccctct cctggcctcg
     2641 cgagatccct ctcgttaagg gaggcaagcc cgacgtcgtc cagattgtcc gcaactacaa
     2701 cgcctacctt cgggccagcg acgatctgcc taagctgttc atcgagtccg accctgggtt
     2761 cttttccaac gctattgtcg agggagctaa gaagttccct aacaccgagt tcgtgaaggt
     2821 gaagggcctc cacttcctcc aggaggacgc tccagatgaa atgggtaagt acatcaagag
     2881 cttcgtggag cgcgtgctga agaacgagca ggctagcgag cagaagctga tctcagagga
     2941 ggacctgagc ggtctgaggt ctagggccca agccagcaat tctcgagtca tggtctatat
     3001 cggaatcgca atcttcctgt tcgttggctt gttcatgaaa tgatctgcag aagcttctag
     3061 gggatccgcc cctctccctc ccccccccct aacgttactg gccgaagccg cttggaataa
     3121 ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg
     3181 agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc
     3241 gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct
     3301 tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac
     3361 aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc
     3421 cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta
     3481 ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg
     3541 cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaaacgtct aggccccccg
     3601 aaccacgggg acgtggtttt cctttgaaaa acacgatgat aatatggcca caaccatggt
     3661 gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca
     3721 catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc
     3781 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc
     3841 ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc
     3901 cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat
     3961 gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga
     4021 gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca
     4081 gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct
     4141 gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt
     4201 caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat
     4261 caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc
     4321 cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtaagcgg ccgcactcct
     4381 caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa
     4441 taccactgag atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag
     4501 catctgactt ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt
     4561 ttgtgtctct cactcggaag gacatatggg agggcaaatc atttaaaaca tcagaatgag
     4621 tatttggttt agagtttggc aacatatgcc atatgctggc tgccatgaac aaaggtggct
     4681 ataaagaggt catcagtata tgaaacagcc ccctgctgtc cattccttat tccatagaaa
     4741 agccttgact tgaggttaga ttttttttat attttgtttt gtgttatttt tttctttaac
     4801 atccctaaaa ttttccttac atgttttact agccagattt ttcctcctct cctgactact
     4861 cccagtcata gctgtccctc ttctcttatg aagatccctc gacctgcagc ccaagcttgg
     4921 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     4981 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     5041 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagcgga
     5101 tccgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     5161 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     5221 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     5281 aggcctaggc ttttgcaaaa agctaacttg tttattgcag cttataatgg ttacaaataa
     5341 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     5401 ttgtccaaac tcatcaatgt atcttatcat gtctggatcc gctgcattaa tgaatcggcc
     5461 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     5521 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     5581 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5641 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     5701 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5761 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5821 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac
     5881 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5941 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     6001 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     6061 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     6121 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     6181 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6241 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     6301 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     6361 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6421 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6481 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6541 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6601 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6661 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6721 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6781 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6841 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6901 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6961 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     7021 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     7081 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     7141 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7201 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7261 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     7321 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     7381 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctg
//
