LOCUS       sequence-322791-        5398 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP tagged TNFa in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5398)
  AUTHORS   Sun X, Mahajan D, Chen B, Song Z, Lu L
  TITLE     A quantitative study of the Golgi retention of glycosyltransferases.
  JOURNAL   J Cell Sci. 2021 Oct 15;134(20). pii: 272560. doi: 
            10.1242/jcs.258564. Epub 2021 Oct 21.
  PUBMED    34533190
REFERENCE   2  (bases 1 to 5398)
  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 Cell 
            Sci."; date: "2021-10-15"; volume: "134(20). pii: 272560. doi"; 
            pages: " 10.1242/jcs.258564. Epub 2021 Oct 21"
COMMENT     Sequence Label: TNFa-GFP
FEATURES             Location/Qualifiers
     source          1..5398
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        92..395
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        396..599
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     550..570
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1370..1379
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1376..1378,1379..1381,1382..2095)
                     /codon_start=1
                     /product="monomeric GFP variant optimized for use in 
                     oxidizing environments such as the secretory pathway 
                     (Costantini et al., 2015)"
                     /label=moxGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFISTTGKLPVPWPTLVTTLTYGVQSFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(1421..1442)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    2217..2338
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2254..2273)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2308..2327
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2345..2800)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2482..2503)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2694..2713
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2827..2931
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2933..3290
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2933..3124
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2956..2976)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3141..3276
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3325..4119
                     /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             3325..4119
                     /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(3379..3398)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3989..4008
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4307..4326)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4351..4398
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4727..5315
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5216..5235
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttctgtggat aaccgtatta ccgccatgca ttagttatta atagtaatca attacggggt
       61 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
      121 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
      181 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
      241 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
      301 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
      361 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
      421 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
      481 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
      541 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg
      601 gtttagtgaa ccgtcagatc cgctagcgct accggactca gatctcgagg ccaccatgtc
      661 taccgagtct atgattaggg acgtggaact ggctgaggag gcactgccca aaaaaaccgg
      721 cggaccacag ggctctagga gatgtctgtt tctgtctctg ttctcttttc tcatcgtggc
      781 tggcgctaca acactcttct gtctgctcca tttcggcgtg attggaccac agcgagagga
      841 atttccccgg gatctgtcac tcatttcacc actggcacag gctgtccgat cttcatctcg
      901 gactccatcc gacaaacctg tcgcccatgt cgtcgccaac ccacaggccg agggccagct
      961 ccagtggctc aataggaggg caaacgctct gctcgccaat ggcgtggaac tccgggataa
     1021 ccagctcgtc gtgcctagtg agggactgta cctcatctac tcccaggtgc tgtttaaggg
     1081 ccagggatgt ccttctacac atgtgctgct cacacacaca atttcacgga tcgccgtgtc
     1141 ttaccagact aaagtcaatc tgctctctgc catcaaatcc ccatgtcagc gggaaacacc
     1201 tgagggcgct gaggctaaac cttggtacga acccatctac ctcggaggcg tgttccagct
     1261 ggagaagggc gatagactga gtgctgagat caatcgaccc gactacctcg actttgccga
     1321 atctggccag gtctactttg gcatcattgc tctgcgggat ccaccggtcg ccaccatggt
     1381 gtccaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     1441 cgtaaacggc cacaagttct ccgtgcgggg cgagggcgag ggcgatgcca ccaacggcaa
     1501 gctgaccctg aagttcatca gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     1561 gaccaccctg acctacggcg tgcagagctt ctcccgctac cccgaccaca tgaagcgcca
     1621 cgacttcttc aagagcgcca tgcccgaagg ctacgtccag gagcgcacca tctccttcaa
     1681 ggacgacggc acctacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     1741 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     1801 ggagtacaac ttcaactccc acaacgtcta tatcaccgcc gacaagcaga agaacggcat
     1861 caaggccaac ttcaagatcc gccacaacgt ggaggacggc tccgtgcagc tcgccgacca
     1921 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     1981 gtccacccag tccaagctgt ccaaagaccc caacgagaag cgcgatcaca tggtccttct
     2041 ggaattcgtg accgccgccg ggatcactca cggcatggac gagctgtaca agtaagcggc
     2101 cgcgactcta gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa
     2161 acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact
     2221 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     2281 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaag
     2341 gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     2401 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     2461 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     2521 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     2581 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     2641 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     2701 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     2761 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga
     2821 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     2881 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tcctgaggcg
     2941 gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     3001 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     3061 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     3121 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     3181 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     3241 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa
     3301 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     3361 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     3421 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     3481 ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg
     3541 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     3601 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     3661 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     3721 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     3781 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     3841 aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     3901 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     3961 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     4021 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     4081 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     4141 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     4201 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     4261 gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg
     4321 agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg
     4381 gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata
     4441 ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc
     4501 ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca
     4561 tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     4621 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     4681 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4741 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4801 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4861 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4921 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4981 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     5041 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     5101 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     5161 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     5221 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     5281 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     5341 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctga
//
