LOCUS       sequence-323471-        5523 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP tagged Tac with mutated five O-glycosylation sites in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5523)
  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 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: "J Biol 
            Chem. 2020 Aug 21. pii: RA120.014476. doi: 
            10.1074/jbc.RA120.014476."
COMMENT     Sequence Label: GFP-Tac(5A)
FEATURES             Location/Qualifiers
     source          1..5523
                     /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    2311..2432
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2348..2367)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2402..2421
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2439..2894)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2576..2597)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2788..2807
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2921..3025
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3027..3384
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3027..3218
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3050..3070)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3235..3370
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3419..4213
                     /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             3419..4213
                     /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(3473..3492)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4083..4102
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4401..4420)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4445..4492
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4821..5409
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5310..5329
                     /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 cgaggagctc tgtgacgatg acccgccaga gatcccacac gccacattca
     1441 aagccatggc ctacaaggaa ggaaccatgt tgaactgtga atgcaagaga ggtttccgca
     1501 gaataaaaag cgggtcactc tatatgctct gtacaggaaa ctctagccac tcgtcctggg
     1561 acaaccaatg tcaatgcaca agctctgcca ctcggaacac aacgaaacaa gtgacacctc
     1621 aacctgaaga acagaaagaa aggaaaacca cagaaatgca aagtccaatg cagccagtgg
     1681 accaagcgag ccttccaggt cactgcaggg aacctccacc atgggaaaat gaagccacag
     1741 agagaattta tcatttcgtg gtggggcaga tggtttatta tcagtgcgtc cagggataca
     1801 gggctctaca cagaggtcct gctgagagcg tctgcaaaat gacccacggg aagacaaggt
     1861 ggacccagcc ccagctcata tgcacaggtg aaatggagac cagtcagttt ccaggtgaag
     1921 agaagcctca ggcaagcccc gaaggccgtc ctgagagtga gacttcctgc ctcgtcacaa
     1981 cagcagattt tcaaatacag gcagaaatgg ctgcagccat ggagacgtcc atatttgcag
     2041 cagagtacca ggtagcagtg gccggctgtg ttttcctgct gatcagcgtc ctcctcctga
     2101 gtgggctcac ctggcagcgg agacagagga agagtagaag aacaatctag gaattctgca
     2161 gtcgacggta ccgcgggccc gggatccacc ggatctagat aactgatcat aatcagccat
     2221 accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg
     2281 aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac
     2341 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     2401 tgtggtttgt ccaaactcat caatgtatct taacgcgtaa attgtaagcg ttaatatttt
     2461 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     2521 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     2581 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     2641 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     2701 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     2761 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     2821 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     2881 gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     2941 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     3001 tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt
     3061 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3121 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3181 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     3241 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     3301 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     3361 tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat
     3421 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     3481 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     3541 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     3601 agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     3661 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     3721 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     3781 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     3841 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     3901 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg
     3961 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     4021 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     4081 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     4141 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     4201 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     4261 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     4321 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     4381 caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct
     4441 atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc
     4501 ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa
     4561 tacgcccgcg tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg
     4621 ctcgcagcca acgtcggggc ggcaggccct gccatagcct caggttactc atatatactt
     4681 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     4741 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     4801 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     4861 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     4921 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     4981 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     5041 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     5101 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     5161 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     5221 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     5281 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     5341 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     5401 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     5461 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg
     5521 cat
//
