LOCUS       sequence-429707-        6601 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Synchronize trafficking of TNFalpha from the ER (RUCH system).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6601)
  AUTHORS   Bottone S, Joliot O, Cakil ZV, El Hajji L, Rakotoarison LM, 
            Boncompain G, Perez F, Gautier A
  TITLE     A fluorogenic chemically induced dimerization technology for 
            controlling, imaging and sensing protein proximity.
  JOURNAL   Nat Methods. 2023 Aug 28. doi: 10.1038/s41592-023-01988-8.
  PUBMED    37640938
REFERENCE   2  (bases 1 to 6601)
  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: "Nat 
            Methods. 2023 Aug 28. doi: 10.1038/s41592-023-01988-8."
COMMENT     Sequence Label: pFP5370 ss-FIREmate-HA- KDEL-IVS-IRES- TNF-SBP...
FEATURES             Location/Qualifiers
     source          1..6601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1431..1457
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     intron          1550..1779
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     1762..1781
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(1851..2425,2426..2428,2429..2437)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2018..2035)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2245..2264
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3195..3308
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     CDS             3318..4025
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3463..3481)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3731..3750
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4221..4342
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4258..4277)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4444..4460)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4444..4460)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4457..4479)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4468..4484
                     /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(4492..4498,4499..4516,4517..4522))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4537..4558
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4675..4692)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4846..5434)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4926..4945)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5605..6396,6397..6465))
                     /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     6228..6247
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6466..6570)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat cgatatctgc ggcctagcta gcgcttaagg cctgttaacc ggtatggatg
      961 tatgcgtccg tcttgccctg tggctcctct ggggactcct cctgcaccag ggccagagcc
     1021 tcagccatag tcacagtgag aaggcgacag gaaccagctc gggcgccggt ttaaacgagc
     1081 atgttgcctt tggcagtgag gacatcgaga acactctggc caatatggac gacgaacaac
     1141 tggataggtt ggcctttggc gtaattcagc tcgatggtga cgggaatatc ctgctgtaca
     1201 atgctgctga aggggacatc actggcagag atcccaaaca ggtgattggg aagaacttct
     1261 tcaaggatgt tgcacctgga acggatactc ccgagtttta cggcaaattc aaggaaggcg
     1321 cagcgtcagg gaatctgaac accatgttcg aatggacgat accgacaagc aggggaccaa
     1381 ccaaggtcaa ggtgcacttg aagaaagccc tttccggagg tggcggaagt tatccttatg
     1441 acgtaccaga ctacgcactc ggaccgaaag atgaactgtg agcggccgca tagataactg
     1501 atccagtgtg ctggaattaa ttcgctgtct gcgagggcca gctgttgggg tgagtactcc
     1561 ctctcaaaag cgggcatgac ttctgcgcta agattgtcag tttccaaaaa cgaggaggat
     1621 ttgatattca cctggcccgc ggtgatgcct ttgagggtgg ccgcgtccat ctggtcagaa
     1681 aagacaatct ttttgttgtc aagcttgagg tgtggcaggc ttgagatctg gccatacact
     1741 tgagtgacaa tgacatccac tttgcctttc tctccacagg tgtccactcc caggtccaac
     1801 tgcaggtcga gcatgcatct agggcggcca attccgcccc tctccccccc cccctctccc
     1861 tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc
     1921 tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc
     1981 cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt
     2041 ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct
     2101 gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa
     2161 aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt
     2221 tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag
     2281 gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt
     2341 acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt
     2401 ttcctttgaa aaacacgatg ataagcttgc cacaacccgg gaggcgcgcc atgtctaccg
     2461 agtctatgat tagggacgtg gaactggctg aggaggcact gcccaaaaaa accggcggac
     2521 cacagggctc taggagatgt ctgtttctgt ctctgttctc ttttctcatc gtggctggcg
     2581 ctacaacact cttctgtctg ctccatttcg gcgtgattgg accacagcga gaggaatttc
     2641 cccgggatct gtcactcatt tcaccactgg cacaggctgt ccgatcttca tctcggactc
     2701 catccgacaa acctgtcgcc catgtcgtcg ccaacccaca ggccgagggc cagctccagt
     2761 ggctcaatag gagggcaaac gctctgctcg ccaatggcgt ggaactccgg gataaccagc
     2821 tcgtcgtgcc tagtgaggga ctgtacctca tctactccca ggtgctgttt aagggccagg
     2881 gatgtccttc tacacatgtg ctgctcacac acacaatttc acggatcgcc gtgtcttacc
     2941 agactaaagt caatctgctc tctgccatca aatccccatg tcagcgggaa acacctgagg
     3001 gcgctgaggc taaaccttgg tacgaaccca tctacctcgg aggcgtgttc cagctggaga
     3061 agggcgatag actgagtgct gagatcaatc gacccgacta cctcgacttt gccgaatctg
     3121 gccaggtcta ctttggcatc attgctctgg gcctcgaggg ccgagctcat ggcgcaccta
     3181 ggcctttgaa ttccatggac gagaagacca ctggttggcg aggtggacac gttgttgaag
     3241 gactggctgg ggaacttgaa caacttcgtg cacgactgga gcatcaccca caaggtcaac
     3301 gtgaaccacc tgcaggtatg gtgagcaagg gcgaggagga taacatggcc atcatcaagg
     3361 agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg
     3421 agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca
     3481 agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg tacggctcca
     3541 aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg
     3601 gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg
     3661 actcctccct acaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc
     3721 cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc tccgagcgga
     3781 tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag ctgaaggacg
     3841 gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc gtgcagctgc
     3901 ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag gactacacca
     3961 tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg gacgagctgt
     4021 acaagggccg gccatctaga ggcggcggct ccggtgacag atattgggtc tttgtgaaac
     4081 gggtgctcga gtgattaatt aactctagat aactgatcat aatcagccat accacatttg
     4141 tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa
     4201 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca
     4261 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     4321 ccaaactcat caatgtatct taacgcgtcg agtgcattct agttgtggtt tgtccaaact
     4381 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     4441 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4501 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     4561 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4621 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     4681 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     4741 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4801 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4861 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4921 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4981 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5041 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5101 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5161 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5221 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5281 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5341 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5401 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5461 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5521 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5581 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5641 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     5701 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     5761 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     5821 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     5881 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     5941 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6001 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6061 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6121 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6181 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6241 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6301 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6361 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6421 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6481 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6541 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     6601 c
//
