LOCUS       sequence-444081-        6812 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient expression of human Eps15 with mGFP tag in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6812)
  AUTHORS   Zhao Y, Caron C, Chan YY, Lee CK, Xu X, Zhang J, Masubuchi T, Wu C, 
            Bui JD, Hui E
  TITLE     cis-B7:CD28 interactions at invaginated synaptic membranes provide 
            CD28 co-stimulation and promote CD8(+) T cell function and 
            anti-tumor immunity.
  JOURNAL   Immunity. 2023 Jun 13;56(6):1187-1203.e12. doi: 
            10.1016/j.immuni.2023.04.005. Epub 2023 May 8.
  PUBMED    37160118
REFERENCE   2  (bases 1 to 6812)
  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"; doi: 
            "10.1016/j.immuni.2023.04.005"; journalName: "Immunity"; date: 
            "2023-06-13- 13"; volume: "56"; issue: "6"; pages: "1187-1203.e12"
COMMENT     Sequence Label: pcDNA-mGFP-Eps15DN
FEATURES             Location/Qualifiers
     source          1..6812
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        68..371
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        372..575
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     526..546
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(627..629,630..632,633..1343)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(672..693)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(933..952)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1280..1301
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1345..1397
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    3607..3728
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3644..3663)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3698..3717
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3735..4190)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3872..3893)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4084..4103
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4217..4321
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4323..4680
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4323..4514
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4346..4366)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4531..4666
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4715..5509
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4715..5509
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4769..4788)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5379..5398
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5697..5716)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5741..5788
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6117..6705
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6606..6625
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgcattag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
       61 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      121 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
      181 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      241 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
      301 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      361 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      421 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      481 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      541 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      601 agcgctaccg gactcagatc acgcgtatgg tgagcaaggg cgaggagctg ttcaccgggg
      661 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
      721 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
      781 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
      841 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
      901 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
      961 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     1021 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     1081 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     1141 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     1201 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccaagctg agcaaagacc
     1261 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     1321 tcggcatgga cgagctgtac aagtctcgag ctcaagcttc gaattctgca gtcgacggta
     1381 ccgcgggccc gggatccatg gctgcggcgg cccagctctc cctgacacag ttatcaagtg
     1441 ggaatcctgt atatgaaaaa tactatagac aggttgatac aggcaatact ggaagggtgt
     1501 tggcttctga tgctgctgct ttcctgaaaa aatcagggct tccagacttg atacttggaa
     1561 agatttggga tttagccgac acagatggca aaggtatcct gaacaaacaa gaattctttg
     1621 ttgctttgcg tcttgtggca tgtgcccaga atggattgga agtttcacta agtagtttgg
     1681 gcattgatcc tcctcacgtt cttactcctg aaatgattcc accatcagac agggccagtt
     1741 tacaaaagaa catcatagga tcaagtcctg ttgcagattt ctctgctatt aaggaactag
     1801 atactcttaa caatgaaata gttgacctac agagggaaaa gaataatgtg gaacaggacc
     1861 ttaaggagaa ggaagatact attaaacaga ggacaagtga ggttcaggat cttcaagatg
     1921 aagttcaaag ggagaatact aatctgcaaa aactacaggc ccagaaacag caggtacagg
     1981 aactccttga tgaactggat gagcagaaag cccagctgga ggagcaactc aaggaagtca
     2041 gaaagaaatg tgctgaggag gcccaactga tctcttctct gaaagctgaa ttaactagtc
     2101 aggaatcgca gatctccact tacgaagaag aattggcaaa agctagagaa gagctgagcc
     2161 gtctacagca agaaacagca gaattggagg agagtgtaga gtcagggaag gctcagttgg
     2221 aacctcttca gcagcaccta caagattcac aacaggaaat tagttcaatg caaatgaaac
     2281 tgatggaaat gaaagatttg gaaaatcata atagtcagtt aaattggtgc agtagcccac
     2341 acagcattct tgtaaacgga gctacagatt attgcagcct cagcaccagc agcagtgaaa
     2401 cagccaacct taatgaacat gttgaaggcc agagcaacct agagtctgag cccatacacc
     2461 aggaatctcc agcaagaagt agtcctgaac tactgccttc tggtgtgact gatgaaaatg
     2521 aggtgactac agctgttact gaaaaagttt gttctgaact cgacaataat agacattcaa
     2581 aagaggaaga tccatttaat gtagactcaa gttcgctgac aggtccagtt gcagatacaa
     2641 acttggattt tttccagtct gatccttttg ttggcagtga tcctttcaag gatgatcctt
     2701 ttggaaaaat cgatccattt ggtggtgatc ctttcaaagg ttcagatcca tttgcatcag
     2761 actgtttctt caggcaatct actgatcctt ttgccacttc aagcactgac cctttcagtg
     2821 cagccaacaa tagcagtatt acatcggtag aaacgttgaa gcacaatgat ccttttgctc
     2881 ctggtggaac agttgttgca gcaagcgatt cagccacaga cccctttgct tctgtttttg
     2941 ggaatgaatc atttggaggt ggatttgctg acttcagcac attgtcaaag gtcaacaatg
     3001 aagatccttt tcgttcagcc acatcgagct ctgtcagcaa cgtagtgatt acaaaaaatg
     3061 tatttgagga aacatcggtc aaaagtgaag atgaaccccc agcactgcca ccaaagatcg
     3121 gaactccaac aagaccctgc cctctaccac ctgggaaaag atccatcaac aaattggatt
     3181 ctcctgatcc ctttaaactg aatgatccat ttcagccttt cccaggcaac gatagcccca
     3241 aagaaaaaga tcctgaaata ttttgtgatc cattcacttc tgctactacc actaccaata
     3301 aagaggctga tccaagcaat tttgccaact tcagtgctta tccctctgaa gaagatatga
     3361 tcgaatgggc caagagggaa agtgagagag aggaagagca gaggcttgcc cgactaaatc
     3421 agcaggaaca agaagactta gaactggcta ttgcactcag caaatctgag atatcagaag
     3481 cataagcggc cgcgactcta gatcataatc agccatacca catttgtaga ggttttactt
     3541 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     3601 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3661 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     3721 gtatcttaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     3781 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     3841 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     3901 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     3961 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     4021 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     4081 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     4141 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac
     4201 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     4261 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     4321 tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     4381 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     4441 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     4501 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     4561 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     4621 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     4681 gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     4741 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     4801 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     4861 caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg
     4921 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     4981 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     5041 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     5101 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     5161 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     5221 actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg
     5281 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     5341 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     5401 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     5461 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     5521 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     5581 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     5641 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa
     5701 acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat
     5761 aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac
     5821 tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc
     5881 ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca
     5941 ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt
     6001 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6061 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     6121 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     6181 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     6241 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     6301 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     6361 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     6421 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     6481 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     6541 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     6601 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     6661 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     6721 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     6781 tcccctgatt ctgtggataa ccgtattacc gc
//
