LOCUS       sequence-309468-        6810 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses mCherry-pBirA-HA/biochemical assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6810)
  AUTHORS   Kwak C, Shin S, Park JS, Jung M, Nhung TTM, Kang MG, Lee C, Kwon TH,
            Park SK, Mun JY, Kim JS, Rhee HW
  TITLE     Contact-ID, a tool for profiling organelle contact sites, reveals 
            regulatory proteins of mitochondrial-associated membrane formation.
  JOURNAL   Proc Natl Acad Sci U S A. 2020 May 15. pii: 1916584117. doi: 
            10.1073/pnas.1916584117.
  PUBMED    32414919
REFERENCE   2  (bases 1 to 6810)
  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: "Proc Natl 
            Acad Sci U S A. 2020 May 15. pii: 1916584117. doi: 
            10.1073/pnas.1916584117."
COMMENT     Sequence Label: pCDNA5/mCherry-pBirA-HA
FEATURES             Location/Qualifiers
     source          1..6810
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    586..604
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    607..625
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     635..659
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             762..1469
                     /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(907..925)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1175..1194
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             join(1482..1829,1830..1832,1833..2441)
                     /codon_start=1
                     /product="promiscuous R118G mutant of the E. coli biotin 
                     protein ligase (Cronan, 2005)"
                     /label=BirA(R118G)
                     /note="also known as BirA* or BioID"
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGGRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKL
                     GADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGW
                     ITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGD
                     KEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             2445..2471
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2528..2545)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2534..2758
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2891..2910)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3042..3089
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3097..4119
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3097..4119
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4249..4370
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4286..4305)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4340..4359
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4419..4435)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4419..4435)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4432..4454)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4443..4459
                     /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(4467..4473,4474..4491,4492..4497))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4512..4533
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4650..4667)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4821..5409)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4901..4920)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5580..6371,6372..6440))
                     /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     6203..6222
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6441..6545)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6620..6639)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata
      601 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
      661 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      721 tccggactct agcgtttaaa cttaagcttg gtaccatcga tatggtgagc aagggcgagg
      781 aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg
      841 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
      901 agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt
      961 cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact
     1021 acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
     1081 gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg
     1141 tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg
     1201 gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca
     1261 agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca
     1321 aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca
     1381 cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact
     1441 ccaccggcgg catggacgag ctgtacaagg aattcggatc caaggacaac accgtgcccc
     1501 tgaagctgat cgccctgctg gccaacggcg agttccactc tggcgagcag ctgggagaga
     1561 ccctgggaat gagcagagcc gccatcaaca agcacatcca gacactgaga gactggggag
     1621 tggacgtgtt caccgtgcct ggcaagggct acagcctgcc tgagcctatc cagctgctga
     1681 acgccaagca gatcctggga cagctggatg gcggaagcgt ggccgtgctg cctgtgatcg
     1741 actccaccaa tcagtacctg ctggacagaa tcggagagct gaagtccggc gacgcctgca
     1801 tcgccgagta ccagcaggct ggcagaggag gcagaggacg gaagtggttc agcccattcg
     1861 gagccaacct gtacctgtcc atgttctgga gactggagca gggacctgct gctgccatcg
     1921 gactgagtct ggtgatcgga atcgtgatgg ccgaggtgct gagaaagctg ggagccgaca
     1981 aggtgagagt gaagtggcct aatgacctgt acctccagga ccgcaagctg gctggcatcc
     2041 tggtggagct gacaggcaag acaggcgatg ccgctcagat cgtgatcgga gccggaatca
     2101 acatggccat gagaagagtg gaggagagcg tggtgaacca gggctggatc accctgcagg
     2161 aggctggcat caacctggac cggaacaccc tggccgccat gctgatcaga gagctgagag
     2221 ccgctctgga gctgttcgag caggagggac tggctcctta cctgagcaga tgggagaagc
     2281 tggacaactt catcaacaga cctgtgaagc tgatcatcgg cgacaaggaa atcttcggca
     2341 tctccagagg aatcgacaag cagggagctc tgctgctgga gcaggacgga atcatcaagc
     2401 cctggatggg cggagaaatc tccctgagaa gcgcagagaa ggcttaccct tacgatgtac
     2461 cggattacgc agcggccgct agggcgcgcc ctcgagtcta gagggcccgt ttaaacccgc
     2521 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     2581 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     2641 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     2701 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     2761 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     2821 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2881 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2941 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3001 gaccccaaaa aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt
     3061 tcctattctc tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg
     3121 tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg
     3181 gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg
     3241 gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg
     3301 gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat
     3361 tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc
     3421 gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag
     3481 acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat
     3541 ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc
     3601 gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc
     3661 gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc
     3721 cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc
     3781 gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc
     3841 gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt
     3901 ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg
     3961 cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc
     4021 gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga
     4081 aaccgacgcc ccagcactcg tccgagggca aaggaatagc acgtactacg agatttcgat
     4141 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     4201 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     4261 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4321 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     4381 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     4441 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4501 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4561 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4621 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4681 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4741 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4801 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4861 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4921 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4981 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5041 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5101 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5161 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5221 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5281 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5341 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5401 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5461 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5521 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5581 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5641 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5701 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5761 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5821 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5881 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5941 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6001 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6061 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6121 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6181 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6241 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6301 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6361 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6421 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6481 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6541 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc
     6601 ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
     6661 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
     6721 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
     6781 ttcgcgatgt acgggccaga tatacgcgtt
//
