LOCUS       sequence-417561-        6674 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TRAM1 fused with 2Strep in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6674)
  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 6674)
  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: TRAM1-2Strep
FEATURES             Location/Qualifiers
     source          1..6674
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(36..55)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        227..606
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        607..810
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     761..781
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     855..874
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        855..873
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2036..2045
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2040..2049
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2057..2080
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2117..2140
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2165..2188
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(2219..2237)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2220..2237)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2257..2274)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2263..2487
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2533..2961
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2620..2639)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2830..2851
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2970..2990)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2975..3304
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3155..3290
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3217..3236
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3371..4165
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3425..3444)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4035..4054
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4339..4460
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4376..4395)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4430..4449
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4509..4525)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4509..4525)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4522..4544)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4533..4549
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4557..4563,4564..4581,4582..4587))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4602..4623
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4740..4757)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4911..5499)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4991..5010)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5670..6461,6462..6530))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6293..6312
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6531..6635)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag
       61 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa
      121 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt
      181 aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg
      241 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
      301 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      361 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      421 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      481 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      541 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      601 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      661 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      721 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      781 gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc cactgcttac
      841 tggcttatcg aaattaatac gactcactat agggagaccc aagcttggta ccgagctcgg
      901 atccatggcg attcgcaaga aaagcaccaa gagcccccca gtgctgagcc acgaattcgt
      961 cctgcagaat cacgcggaca tcgtctcctg tgtggcgatg gtcttcctgc tggggctcat
     1021 gtttgagata acggcaaaag cttctatcat ttttgttact cttcagtaca atgtcaccct
     1081 cccagcaaca gaagaacaag ctactgaatc agtgtccctt tattactatg gcatcaaaga
     1141 tttggctact gttttcttct acatgctagt ggcgataatt attcatgccg taattcaaga
     1201 gtatatgttg gataaaatta acaggcgaat gcacttctcc aaaacaaaac acagcaagtt
     1261 taatgaatct ggtcagctta gtgcgttcta cctttttgcc tgtgtttggg gcacattcat
     1321 tctcatctct gaaaactaca tctcagaccc aactatctta tggagggctt atccccataa
     1381 cctgatgaca tttcaaatga agtttttcta catatcacag ctggcttact ggcttcatgc
     1441 ttttcctgaa ctctacttcc agaaaaccaa aaaagaagat attcctcgtc agcttgtcta
     1501 cattggtctt tacctcttcc acattgctgg agcttacctt ttgaacttga atcatctagg
     1561 acttgttctt ctggtgctac attattttgt tgaatttctt ttccacattt cccgcctgtt
     1621 ttattttagc aatgaaaagt atcagaaagg attttctctg tgggcagttc tttttgtttt
     1681 gggaagactt ctgactttaa ttctttcagt actgactgtt ggttttggcc ttgcaagagc
     1741 agaaaatcag aagctggatt tcagtactgg aaacttcaat gtgttagctg ttagaatcgc
     1801 tgttctggca tccatttgcg ttactcaggc atttatgatg tggaagttca ttaattttca
     1861 gcttcgaagg tggagggaac attctgcttt tcaggcacca gctgtgaaga agaaaccaac
     1921 agtaactaaa ggcagatctt ctaaaaaagg aacagaaaat ggtgtgaatg gaacattaac
     1981 ttcaaatgta gcagactctc cccggaataa aaaagagaaa tcttcagaat tcgccgccac
     2041 catggcggcc gccagctgga gccaccctca gttcgagaag ggaggaggaa gcggcggagg
     2101 cagcggagga ggaagctgga gccacccgca gttcgagaaa tgactcgagc atgcatctag
     2161 aggctggagc cacccgcagt tcgagaaatg actcgagcat gcatctagag ggccctattc
     2221 tatagtgtca cctaaatgct agagctcgct gatcagcctc gactgtgcct tctagttgcc
     2281 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     2341 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     2401 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     2461 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta
     2521 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     2581 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     2641 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     2701 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     2761 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     2821 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     2881 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     2941 aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc
     3001 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     3061 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3121 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     3181 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     3241 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     3301 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg
     3361 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     3421 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     3481 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     3541 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     3601 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     3661 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     3721 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     3781 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     3841 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg
     3901 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     3961 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     4021 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     4081 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     4141 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     4201 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     4261 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     4321 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     4381 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     4441 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     4501 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     4561 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     4621 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4681 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     4741 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     4801 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     4861 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4921 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4981 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5041 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5101 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5161 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     5221 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     5281 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     5341 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     5401 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     5461 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5521 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     5581 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     5641 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     5701 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     5761 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     5821 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     5881 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     5941 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     6001 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6061 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6121 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6181 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     6241 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     6301 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     6361 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     6421 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     6481 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     6541 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     6601 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     6661 gacgtcgacg gatc
//
