LOCUS       sequence-309462-        5978 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses flag-pbira(1-g78)-sec61b/biochemical assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5978)
  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 5978)
  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)-sec61b
FEATURES             Location/Qualifiers
     source          1..5978
                     /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             351..641
                     /codon_start=1
                     /gene="SEC61B"
                     /product="human Sec61-beta subunit of the translocon in the
                     endoplasmic reticulum"
                     /label=Sec61-beta
                     /translation="MPGPTPSGTNVGSSGRSPSKAVAARAAGSTVRQRKNASCGTRSAG
                     RTTSAGTGGMWRFYTEDSPGLKVGPVPVLVMSLLFIASVFMLHIWGKYTRS"
     promoter        complement(669..687)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(670..687)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(707..724)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    713..937
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      983..1411
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1070..1089)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1280..1301
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1420..1440)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1425..1754
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1605..1740
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1667..1686
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1821..2615
                     /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(1875..1894)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2485..2504
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2789..2910
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2826..2845)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2880..2899
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2959..2975)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2959..2975)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2972..2994)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2983..2999
                     /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(3007..3013,3014..3031,3032..3037))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3052..3073
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3190..3207)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3361..3949)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3441..3460)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4120..4911,4912..4980))
                     /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     4743..4762
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4981..5085)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5160..5179)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5351..5730
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5731..5934
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5885..5905
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 taatacgact cactataggg agacccaagc ttgcggccgc caccatggac tacaaggatg
       61 acgacgataa gaaggacaac accgtgcccc tgaagctgat cgccctgctg gccaacggcg
      121 agttccactc tggcgagcag ctgggagaga ccctgggaat gagcagagcc gccatcaaca
      181 agcacatcca gacactgaga gactggggag tggacgtgtt caccgtgcct ggcaagggct
      241 acagcctgcc tgagcctatc cagctgctga acgccaagca gatcctggga cagctggatg
      301 gcgggggtgc gtcaggcggc agcggatctg gaccggtcgc caccgaattc atgcctggtc
      361 cgacccccag tggcactaac gtgggatcct cagggcgctc tcccagcaaa gcagtggccg
      421 cccgggcggc gggatccact gtccggcaga ggaaaaatgc cagctgtggg acaaggagtg
      481 caggccgcac aacctcggca ggcaccgggg ggatgtggcg attctacaca gaagattcac
      541 ctgggctcaa agttggccct gttccagtat tggttatgag tcttctgttc atcgcttctg
      601 tatttatgtt gcacatttgg ggcaagtaca ctcgttcgtg actcgagcat gcatctagag
      661 ggccctattc tatagtgtca cctaaatgct agagctcgct gatcagcctc gactgtgcct
      721 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
      781 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
      841 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
      901 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
      961 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     1021 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     1081 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     1141 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     1201 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     1261 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     1321 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     1381 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt
     1441 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     1501 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     1561 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     1621 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     1681 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     1741 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga
     1801 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     1861 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     1921 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     1981 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg
     2041 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     2101 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     2161 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     2221 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     2281 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     2341 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     2401 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     2461 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     2521 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     2581 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac
     2641 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga
     2701 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     2761 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     2821 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     2881 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     2941 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     3001 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     3061 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     3121 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     3181 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     3241 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     3301 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     3361 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     3421 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     3481 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     3541 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     3601 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     3661 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     3721 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     3781 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     3841 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     3901 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     3961 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     4021 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     4081 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     4141 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     4201 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     4261 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     4321 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     4381 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     4441 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     4501 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     4561 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     4621 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     4681 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     4741 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     4801 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     4861 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     4921 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     4981 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     5041 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     5101 agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt
     5161 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
     5221 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
     5281 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
     5341 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
     5401 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     5461 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     5521 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     5581 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     5641 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     5701 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
     5761 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
     5821 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
     5881 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg
     5941 ctaactagag aacccactgc ttactggctt atcgaaat
//
