LOCUS       sequence-309447-        5951 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses pBirA(1-G78)-FRB/biochemical assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5951)
  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 5951)
  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: pCDNA3/flag-pBirA(1-G78)-FRB
FEATURES             Location/Qualifiers
     source          1..5951
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      39..48
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             48..71
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             324..602
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     promoter        complement(642..660)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(643..660)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(680..697)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    686..910
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      956..1384
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1043..1062)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1253..1274
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1393..1413)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1398..1727
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1578..1713
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1640..1659
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1794..2588
                     /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(1848..1867)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2458..2477
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2762..2883
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2799..2818)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2853..2872
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2932..2948)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2932..2948)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2945..2967)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2956..2972
                     /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(2980..2986,2987..3004,3005..3010))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3025..3046
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3163..3180)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3334..3922)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3414..3433)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4093..4884,4885..4953))
                     /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     4716..4735
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4954..5058)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5133..5152)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5324..5703
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5704..5907
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5858..5878
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 taatacgact cactataggg agacccaagc ttgcggccgc caccatggac tacaaggatg
       61 acgacgataa gatgaaggac aacaccgtgc ccctgaagct gatcgccctg ctggccaacg
      121 gcgagttcca ctctggcgag cagctgggag agaccctggg aatgagcaga gccgccatca
      181 acaagcacat ccagacactg agagactggg gagtggacgt gttcaccgtg cctggcaagg
      241 gctacagcct gcctgagcct atccagctgc tgaacgccaa gcagatcctg ggacagctgg
      301 atggcggatc catggcttct agaatcctct ggcatgagat gtggcatgaa ggcctggaag
      361 aggcatctcg tttgtacttt ggggaaagga acgtgaaagg catgtttgag gtgctggagc
      421 ccttgcatgc tatgatggaa cggggccccc agactctgaa ggaaacatcc tttaatcagg
      481 cctatggtcg agatttaatg gaggcccaag agtggtgcag gaagtacatg aaatcaggga
      541 atgtcaagga cctcctccaa gcctgggacc tctattatca tgtgttccga cgaatctcaa
      601 agactagtta atgactcgag catgcatcta gagggcccta ttctatagtg tcacctaaat
      661 gctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
      721 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
      781 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
      841 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
      901 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
      961 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     1021 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     1081 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     1141 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     1201 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     1261 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     1321 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     1381 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     1441 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     1501 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     1561 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     1621 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     1681 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     1741 cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     1801 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     1861 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     1921 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     1981 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     2041 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     2101 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     2161 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     2221 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     2281 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     2341 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     2401 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     2461 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     2521 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     2581 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     2641 acgagatttc gattccacca ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     2701 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     2761 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     2821 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     2881 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     2941 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     3001 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     3061 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     3121 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     3181 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     3241 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     3301 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3361 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3421 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     3481 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     3541 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     3601 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     3661 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     3721 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     3781 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     3841 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     3901 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     3961 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     4021 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     4081 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     4141 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     4201 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     4261 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     4321 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     4381 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     4441 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     4501 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     4561 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     4621 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     4681 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     4741 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     4801 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     4861 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     4921 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     4981 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     5041 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
     5101 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     5161 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
     5221 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
     5281 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact
     5341 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
     5401 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
     5461 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
     5521 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
     5581 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
     5641 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
     5701 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
     5761 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
     5821 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
     5881 cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac tgcttactgg
     5941 cttatcgaaa t
//
