LOCUS       sequence-417562-        5816 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TRAM1 fused with EGFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5816)
  AUTHORS   Li J, Gui Q, Liang FX, Sall J, Zhang Q, Duan Y, Dhabaria A, Askenazi
            M, Ueberheide B, Stapleford KA, Pagano M
  TITLE     The REEP5/TRAM1 complex binds SARS-CoV-2 NSP3 and promotes virus 
            replication.
  JOURNAL   J Virol. 2023 Sep 28:e0050723. doi: 10.1128/jvi.00507-23.
  PUBMED    37768083
REFERENCE   2  (bases 1 to 5816)
  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 Virol. 
            2023 Sep 28:e0050723. doi: 10.1128/jvi.00507-23."
COMMENT     Sequence Label: TRAM1-EGFP
FEATURES             Location/Qualifiers
     source          1..5816
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        63..366
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        367..570
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     521..541
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1758..1767
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1764..1766,1767..1769,1770..2483)
                     /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(1809..1830)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2070..2089)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2417..2438
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2606..2727
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2643..2662)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2697..2716
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2734..3189)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2871..2892)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3083..3102
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3216..3320
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3322..3679
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3322..3513
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3345..3365)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3530..3665
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3714..4508
                     /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             3714..4508
                     /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(3768..3787)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4378..4397
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4696..4715)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4740..4787
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5116..5704
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5605..5624
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 attagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
       61 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      121 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      181 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      241 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      301 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      361 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      421 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      481 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      541 gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagcgc
      601 taccggactc agatctcgag atggcgattc gcaagaaaag caccaagagc cccccagtgc
      661 tgagccacga attcgtcctg cagaatcacg cggacatcgt ctcctgtgtg gcgatggtct
      721 tcctgctggg gctcatgttt gagataacgg caaaagcttc tatcattttt gttactcttc
      781 agtacaatgt caccctccca gcaacagaag aacaagctac tgaatcagtg tccctttatt
      841 actatggcat caaagatttg gctactgttt tcttctacat gctagtggcg ataattattc
      901 atgccgtaat tcaagagtat atgttggata aaattaacag gcgaatgcac ttctccaaaa
      961 caaaacacag caagtttaat gaatctggtc agcttagtgc gttctacctt tttgcctgtg
     1021 tttggggcac attcattctc atctctgaaa actacatctc agacccaact atcttatgga
     1081 gggcttatcc ccataacctg atgacatttc aaatgaagtt tttctacata tcacagctgg
     1141 cttactggct tcatgctttt cctgaactct acttccagaa aaccaaaaaa gaagatattc
     1201 ctcgtcagct tgtctacatt ggtctttacc tcttccacat tgctggagct taccttttga
     1261 acttgaatca tctaggactt gttcttctgg tgctacatta ttttgttgaa tttcttttcc
     1321 acatttcccg cctgttttat tttagcaatg aaaagtatca gaaaggattt tctctgtggg
     1381 cagttctttt tgttttggga agacttctga ctttaattct ttcagtactg actgttggtt
     1441 ttggccttgc aagagcagaa aatcagaagc tggatttcag tactggaaac ttcaatgtgt
     1501 tagctgttag aatcgctgtt ctggcatcca tttgcgttac tcaggcattt atgatgtgga
     1561 agttcattaa ttttcagctt cgaaggtgga gggaacattc tgcttttcag gcaccagctg
     1621 tgaagaagaa accaacagta actaaaggca gatcttctaa aaaaggaaca gaaaatggtg
     1681 tgaatggaac attaacttca aatgtagcag actctccccg gaataaaaaa gagaaatctt
     1741 cagcggatcc accggtcgcc accatggtga gcaagggcga ggagctgttc accggggtgg
     1801 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     1861 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     1921 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     1981 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     2041 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     2101 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     2161 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     2221 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     2281 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     2341 ccgtgctgct gcccgacaac cactacctga gcacccagtc caagctgagc aaagacccca
     2401 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     2461 gcatggacga gctgtacaag taaagcggcc gcgactctag atcataatca gccataccac
     2521 atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca
     2581 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata
     2641 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     2701 tttgtccaaa ctcatcaatg tatcttaagg cgtaaattgt aagcgttaat attttgttaa
     2761 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     2821 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     2881 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     2941 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     3001 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     3061 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     3121 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     3181 agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     3241 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     3301 aatattgaaa aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt
     3361 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3421 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3481 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     3541 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     3601 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     3661 aggcctaggc ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg
     3721 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     3781 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     3841 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg
     3901 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     3961 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     4021 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     4081 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     4141 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     4201 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg
     4261 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     4321 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     4381 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     4441 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     4501 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     4561 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     4621 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     4681 tagggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac
     4741 ggcaataaaa agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt
     4801 tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc
     4861 ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc
     4921 agccaacgtc ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat
     4981 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     5041 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     5101 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     5161 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     5221 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     5281 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     5341 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     5401 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     5461 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     5521 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     5581 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     5641 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     5701 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     5761 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgc
//
