LOCUS       sequence-322571-        5618 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of EGFP-tagged proteins under CAG promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5618)
  TITLE     Franz lab unpublished
REFERENCE   2  (bases 1 to 5618)
  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"
COMMENT     Sequence Label: pCAG-MCS-EGFP
FEATURES             Location/Qualifiers
     source          1..5618
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1828..1837
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1834..1836,1837..1839,1840..2553)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1834..1836,1837..1839,1840..2550)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1879..1900)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2140..2159)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2487..2508
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2640..2659)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2705..2760
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2759..2778)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3121..3137)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3121..3137)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3134..3156)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3145..3161
                     /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(3169..3175,3176..3193,3194..3199))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3214..3235
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3294..3489
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3340..3475
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3495..3629
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3532..3551)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3586..3605
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3697..3714)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3868..4456)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3948..3967)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4627..5418,5419..5487))
                     /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     5250..5269
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5488..5592)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggggcgc gccaggsggg gsggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcgctagctc
     1741 gagcgcgcgc gtgtacacgt cttcgaagag ctccttaaga cgcgtatcga tatcaattgc
     1801 gtcgacggca cccgggatcc accggtcgcc accatggtga gcaagggcga ggagctgttc
     1861 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     1921 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     1981 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     2041 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     2101 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     2161 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     2221 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     2281 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     2341 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     2401 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     2461 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     2521 atcactctcg gcatggacga gctgtacaag taaagcggcc gcactcctca ggtgcaggct
     2581 gcctatcaga aggtggtggc tggtgtggcc aatgccctgg ctcacaaata ccactgagat
     2641 ctttttccct ctgccaaaaa ttatggggac atcatgaagc cccttgagca tctgacttct
     2701 ggctaataaa ggaaatttat tttcattgca atagtgtgtt ggaatttttt gtgtctctca
     2761 ctcggaagga catatgggag ggcaaatcat ttaaaacatc agaatgagta tttggtttag
     2821 agtttggcaa catatgccca tatgctggct gccatgaaca aaggttggct ataaagaggt
     2881 catcagtata tgaaacagcc ccctgctgtc cattccttat tccatagaaa agccttgact
     2941 tgaggttaga ttttttttat attttgtttt gtgttatttt tttctttaac atccctaaaa
     3001 ttttccttac atgttttact agccagattt ttcctcctct cctgactact cccagtcata
     3061 gctgtccctc ttctcttatg gagatccctc gacctgcagc ccaagcttgg cgtaatcatg
     3121 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     3181 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     3241 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagcgga tccgcatctc
     3301 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     3361 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     3421 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     3481 ttttgcaaaa agctaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     3541 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     3601 tcatcaatgt atcttatcat gtctggatcc gctgcattaa tgaatcggcc aacgcgcggg
     3661 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3721 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3781 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3841 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3901 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3961 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4021 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4081 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4141 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4201 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4261 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4321 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4381 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4441 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4501 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4561 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4621 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     4681 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     4741 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     4801 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     4861 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     4921 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     4981 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5041 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     5101 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     5161 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     5221 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     5281 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     5341 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     5401 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     5461 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     5521 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5581 aggggttccg cgcacatttc cccgaaaagt gccacctg
//
