LOCUS       sequence-399459-        5523 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express N-terminal AcGFP-fused protein  in mammalian cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5523)
  AUTHORS   Furuta M, Murakami C, Numagami Y, Suzuki R, Sakane F
  TITLE     Diacylglycerol kinase zeta interacts with sphingomyelin synthase 1 
            and sphingomyelin synthase-related protein via different regions.
  JOURNAL   FEBS Open Bio. 2023 May 11. doi: 10.1002/2211-5463.13628.
  PUBMED    37166445
REFERENCE   2  (bases 1 to 5523)
  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: "FEBS Open 
            Bio. 2023 May 11. doi: 10.1002/2211-5463.13628."
COMMENT     Sequence Label: pCAGGS-AcGFP-C
FEATURES             Location/Qualifiers
     source          1..5523
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          663..1679
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1603..1625
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1687..1706
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1733..1742
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1739..2455
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     misc_feature    2456..2521
                     /label=MCS
                     /note="multiple cloning site"
     primer_bind     complement(2545..2564)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2610..2665
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2664..2683)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3026..3042)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3026..3042)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3039..3061)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3050..3066
                     /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(3074..3080,3081..3098,3099..3104))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3119..3140
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3199..3394
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3245..3380
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3400..3534
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3437..3456)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3491..3510
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3602..3619)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3773..4361)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3853..3872)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4532..5323,5324..5392))
                     /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     5155..5174
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5393..5497)
                     /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 gggggnsgcg cgccrggsgg ggsggggsgg ggsgrggggs
      541 ggggsggggs grggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt
      601 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
      661 cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc
      721 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
      781 ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg
      841 aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggggtgcgt
      901 gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc ggctgtgagc
      961 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg agcgcggccg
     1021 ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc gtgcggggtg
     1081 tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa ccccccctgc
     1141 acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc
     1201 gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg
     1261 cggggccgcc tcgggccggg grgggctcgg gggaggggcg cggcggcccc cggagcgccg
     1321 gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg
     1381 cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca
     1441 ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg
     1501 agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct cggggctgtc
     1561 cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg
     1621 tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc
     1681 tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttgccaccat
     1741 ggtgagcaag ggcgccgagc tgttcaccgg catcgtgccc atcctgatcg agctgaatgg
     1801 cgatgtgaat ggccacaagt tcagcgtgag cggcgagggc gagggcgatg ccacctacgg
     1861 caagctgacc ctgaagttca tctgcaccac cggcaagctg cctgtgccct ggcccaccct
     1921 ggtgaccacc ctgagctacg gcgtgcagtg cttctcacgc taccccgatc acatgaagca
     1981 gcacgacttc ttcaagagcg ccatgcctga gggctacatc caggagcgca ccatcttctt
     2041 cgaggatgac ggcaactaca agtcgcgcgc cgaggtgaag ttcgagggcg ataccctggt
     2101 gaatcgcatc gagctgaccg gcaccgattt caaggaggat ggcaacatcc tgggcaataa
     2161 gatggagtac aactacaacg cccacaatgt gtacatcatg accgacaagg ccaagaatgg
     2221 catcaaggtg aacttcaaga tccgccacaa catcgaggat ggcagcgtgc agctggccga
     2281 ccactaccag cagaataccc ccatcggcga tggccctgtg ctgctgcccg ataaccacta
     2341 cctgtccacc cagagcgccc tgtccaagga ccccaacgag aagcgcgatc acatgatcta
     2401 cttcggcttc gtgaccgccg ccgccatcac ccacggcatg gatgagctgt acaagtccgg
     2461 actcagatct cgagctcaag cttcgaattc tgcagtcgac ggtaccgcgg gcccgggatc
     2521 caccggatct agatactgat cagatctttt tccctctgcc aaaaattatg gggacatcat
     2581 gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt
     2641 gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcatttaaa
     2701 acatcagaat gagtatttgg tttagagttt ggcaacatat gcccatatgc tggctgccat
     2761 gaacaaaggt tggctataaa gaggtcatca gtatatgaaa cagccccctg ctgtccattc
     2821 cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt gttttgtgtt
     2881 atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca gatttttcct
     2941 cctctcctga ctactcccag tcatagctgt ccctcttctc ttatggagat ccctcgacct
     3001 gcagcccaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     3061 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     3121 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3181 tgtcgtgcca gcggatccgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     3241 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     3301 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     3361 aggaggcttt tttggaggcc taggcttttg caaaaagcta acttgtttat tgcagcttat
     3421 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     3481 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatccgctgc
     3541 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     3601 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     3661 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     3721 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     3781 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3841 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     3901 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     3961 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4021 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4081 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4141 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4201 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4261 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4321 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4381 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     4441 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     4501 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     4561 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     4621 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     4681 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     4741 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     4801 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     4861 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     4921 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     4981 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     5041 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     5101 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     5161 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     5221 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     5281 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     5341 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     5401 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     5461 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     5521 ctg
//
