LOCUS       sequence-323489-        5412 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP tagged CD8a mutant in mammalian cells. The seven 
            O-glycosylation sites in its stem region are mutated..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5412)
  AUTHORS   Sun X, Tie HC, Chen B, Lu L
  TITLE     Glycans function as a Golgi export signal to promote the 
            constitutive exocytic trafficking.
  JOURNAL   J Biol Chem. 2020 Aug 21. pii: RA120.014476. doi: 
            10.1074/jbc.RA120.014476.
  PUBMED    32826314
REFERENCE   2  (bases 1 to 5412)
  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: "J Biol 
            Chem. 2020 Aug 21. pii: RA120.014476. doi: 
            10.1074/jbc.RA120.014476."
COMMENT     Sequence Label: GFP-CD8a(7A)
FEATURES             Location/Qualifiers
     source          1..5412
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      597..606
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(672..674,675..677,678..1388)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(672..674,675..677,678..1388)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(717..738)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(978..997)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1325..1346
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2200..2321
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2237..2256)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2291..2310
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2328..2783)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2465..2486)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2677..2696
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2810..2914
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2916..3273
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2916..3107
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2939..2959)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3124..3259
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3308..4102
                     /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             3308..4102
                     /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(3362..3381)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3972..3991
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4290..4309)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4334..4381
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4710..5298
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5199..5218
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcca
      601 ccatggattc atacctgctg atgtggggac tgctcacgtt catcatggtg cctggctgcc
      661 aggcaaccgg tatggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
      721 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
      781 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
      841 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
      901 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
      961 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     1021 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     1081 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     1141 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     1201 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     1261 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     1321 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     1381 tgtacaagct cgagagccag ttccgggtgt cgccgctgga tcggacctgg aacctgggcg
     1441 agacagtgga gctgaagtgc caggtgctgc tgtccaaccc gacgtcgggc tgctcgtggc
     1501 tcttccagcc gcgcggcgcc gccgccagtc ccaccttcct cctatacctc tcccaaaaca
     1561 agcccaaggc ggccgagggg ctggacaccc agcggttctc gggcaagagg ttgggggaca
     1621 ccttcgtcct caccctgagc gacttccgcc gagagaacga gggctactat ttctgctcgg
     1681 ccctgagcaa ctccatcatg tacttcagcc acttcgtgcc ggtcttcctg ccagcgaagc
     1741 ccgcagcagc tccagcgccg cgaccaccag caccggcgcc cgcaatcgcg gcacagcccc
     1801 tggcactgcg cccagaggcg tgtcggccag cggcgggggg cgcagtgcac acgagggggc
     1861 tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt ggggtccttc
     1921 tcctgtcact ggttatcacc ctttactgca accacaggaa ccgaagacgt gtttgcaaat
     1981 gtccccggcc tgtggtcaaa tcgggagaca agcccagcct ttcggcgaga tacgtctaag
     2041 aattctgcag tcgacggtac cgcgggcccg ggatccaccg gatctagata actgatcata
     2101 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
     2161 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat
     2221 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     2281 cattctagtt gtggtttgtc caaactcatc aatgtatctt aacgcgtaaa ttgtaagcgt
     2341 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     2401 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     2461 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     2521 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     2581 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     2641 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     2701 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     2761 taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     2821 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     2881 ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg
     2941 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3001 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     3061 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     3121 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     3181 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     3241 ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc
     3301 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     3361 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     3421 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     3481 tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     3541 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     3601 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     3661 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     3721 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     3781 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat
     3841 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     3901 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     3961 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     4021 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     4081 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg
     4141 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     4201 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     4261 ttcttcgccc accctagggg gaggctaact gaaacacgga aggagacaat accggaagga
     4321 acccgcgcta tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt
     4381 cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat
     4441 tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa
     4501 ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca
     4561 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     4621 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     4681 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     4741 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     4801 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     4861 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     4921 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     4981 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     5041 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     5101 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     5161 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     5221 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     5281 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     5341 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     5401 accgccatgc at
//
