LOCUS       sequence-429706-        6226 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Synchronize anterograde and retrograde trafficking of ManII between 
            ER and Golgi (RUCH system).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6226)
  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 6226)
  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: pFP5299 ss-FIREmate-HA- KDEL-IVS-IRES- ManII-S...
FEATURES             Location/Qualifiers
     source          1..6226
                     /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             2826..2936
                     /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="DEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     CDS             2946..3650
                     /codon_start=1
                     /product="photostable monomeric derivative of DsRed (Shaner
                     et al., 2008)"
                     /label=mApple
                     /note="mammalian codon-optimized"
                     /translation="VSKGEENNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEAF
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKVYIKHPADIPDYFKLSFPEGFRWERVMNFE
                     DGGIIHVNQDSSLQDGVFIYKVKLRGTNFPSDGPVMQKKTMGWEASEERMYPEDGALKS
                     EIKKRLKLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     3356..3375
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3846..3967
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3883..3902)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4069..4085)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4069..4085)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4082..4104)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4093..4109
                     /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(4117..4123,4124..4141,4142..4147))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4162..4183
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4300..4317)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4471..5059)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4551..4570)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5230..6021,6022..6090))
                     /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     5853..5872
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6091..6195)
                     /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 atgaagttaa
     2461 gtcgccagtt caccgtgttt ggcagcgcga tcttctgcgt cgtaatcttc tcactctacc
     2521 tgatgctgga caggggtcac ttggactacc ctcggggccc gcgccaggag ggctcctttc
     2581 cgcagggcca gctttcaata ttgcaagaaa agattgacca tttggagcgt ttgctcgctg
     2641 agaacaacga gattatctca aatatcagag actcagtcat caacctgagc gagtctgtgg
     2701 aggacggccc gcgggggtca ccaggcaacg ccagccaagg ctccatccac ctccactcgc
     2761 cacagttggc cctgcaggct gaccccagag actgtttgga tcccaccggt cgccaccgga
     2821 attccgacga gaagaccact ggttggcgag gtggacacgt tgttgaagga ctggctgggg
     2881 aacttgaaca acttcgtgca cgactggagc atcacccaca aggtcaacgt gaaccacctg
     2941 caggtgtgag caagggcgag gagaataaca tggccatcat caaggagttc atgcgcttca
     3001 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg
     3061 gccgccccta cgaggccttt cagaccgcta agctgaaggt gaccaagggt ggccccctgc
     3121 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggtc tacattaagc
     3181 acccagccga catccccgac tacttcaagc tgtccttccc cgagggcttc aggtgggagc
     3241 gcgtgatgaa cttcgaggac ggcggcatta ttcacgttaa ccaggactcc tccctgcagg
     3301 acggcgtgtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg
     3361 taatgcagaa gaagaccatg ggctgggagg cctccgagga gcggatgtac cccgaggacg
     3421 gcgccctgaa gagcgagatc aagaagaggc tgaagctgaa ggacggcggc cactacgccg
     3481 ccgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacatcg
     3541 tcgacatcaa gttggacatc gtgtcccaca acgaggacta caccatcgtg gaacagtacg
     3601 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag ggccggccag
     3661 gcggcggctc cggtgacaga tattgggtct ttgtgaaacg ggtgggcctc gagggccaat
     3721 taattaactc tagataactg atcataatca gccataccac atttgtagag gttttacttg
     3781 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     3841 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     3901 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     3961 tatcttaacg cgtcgagtgc attctagttg tggtttgtcc aaactcatca atgtatctta
     4021 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     4081 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     4141 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     4201 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4261 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4321 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4381 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4441 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4501 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4561 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     4621 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     4681 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     4741 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     4801 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     4861 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     4921 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     4981 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5041 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5101 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5161 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     5221 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     5281 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     5341 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     5401 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     5461 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     5521 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     5581 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     5641 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     5701 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     5761 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     5821 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     5881 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     5941 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     6001 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     6061 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     6121 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     6181 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
