LOCUS       sequence-350118-        7124 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  myc-BioID fused to human Tollip carrying CUE domain mutations cloned
            into the pIRESneo2 plasmid. For generation of stable expressing 
            mammalian cells used for proximity dependent biotin labelling..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7124)
  AUTHORS   Ryan TA, Phillips EO, Collier CL, Jb Robinson A, Routledge D, Wood 
            RE, Assar EA, Tumbarello DA
  TITLE     Tollip coordinates Parkin-dependent trafficking of 
            mitochondrial-derived vesicles.
  JOURNAL   EMBO J. 2020 Jun 2;39(11):e102539. doi: 10.15252/embj.2019102539. 
            Epub 2020 Apr 20.
  PUBMED    32311122
REFERENCE   2  (bases 1 to 7124)
  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: "EMBO J."; 
            date: "2020-06-2"; volume: "39(11):e102539. doi"; pages: " 
            10.15252/embj.2019102539. Epub 2020 Apr 20"
COMMENT     Sequence Label: pIRESneo2 mycBioID-Tollip M240A,F241A
FEATURES             Location/Qualifiers
     source          1..7124
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        101..480
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        481..684
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     635..655
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     729..748
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        729..747
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             815..844
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             join(848..1195,1196..1198,1199..1807)
                     /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"
     intron          2731..2960
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2943..2962
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(3033..3607,3608..3610,3611..3619)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3200..3217)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3427..3446
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3642..4445
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3705..3724)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4315..4334
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4477..4494)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4483..4707
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4754..4773
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4833..4849)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4833..4849)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4846..4868)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4857..4873
                     /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(4881..4887,4888..4905,4906..4911))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4926..4947
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5064..5081)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5235..5823)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5315..5334)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5994..6785,6786..6854))
                     /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     6617..6636
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6855..6959)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7034..7053)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt
       61 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt
      121 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
      181 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      241 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
      301 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
      361 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
      421 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
      481 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
      541 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      601 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      661 tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc ttactggctt
      721 atcgaaatta atacgactca ctatagggag acccaagctt ggtaccgagc tcggatcgat
      781 atctgcggcc tagctagcgc taccggtcac catggaacaa aaactcatct cagaagagga
      841 tctcgacaag gacaacaccg tgcccctgaa gctgatcgcc ctgctggcca acggcgagtt
      901 ccactctggc gagcagctgg gagagaccct gggaatgagc agagccgcca tcaacaagca
      961 catccagaca ctgagagact ggggagtgga cgtgttcacc gtgcctggca agggctacag
     1021 cctgcctgag cctatccagc tgctgaacgc caagcagatc ctgggacagc tggatggcgg
     1081 aagcgtggcc gtgctgcctg tgatcgactc caccaatcag tacctgctgg acagaatcgg
     1141 agagctgaag tccggcgacg cctgcatcgc cgagtaccag caggctggca gaggaggcag
     1201 aggacggaag tggttcagcc cattcggagc caacctgtac ctgtccatgt tctggagact
     1261 ggagcaggga cctgctgctg ccatcggact gagtctggtg atcggaatcg tgatggccga
     1321 ggtgctgaga aagctgggag ccgacaaggt gagagtgaag tggcctaatg acctgtacct
     1381 ccaggaccgc aagctggctg gcatcctggt ggagctgaca ggcaagacag gcgatgccgc
     1441 tcagatcgtg atcggagccg gaatcaacat ggccatgaga agagtggagg agagcgtggt
     1501 gaaccagggc tggatcaccc tgcaggaggc tggcatcaac ctggaccgga acaccctggc
     1561 cgccatgctg atcagagagc tgagagccgc tctggagctg ttcgagcagg agggactggc
     1621 tccttacctg agcagatggg agaagctgga caacttcatc aacagacctg tgaagctgat
     1681 catcggcgac aaggaaatct tcggcatctc cagaggaatc gacaagcagg gagctctgct
     1741 gctggagcag gacggaatca tcaagccctg gatgggcgga gaaatctccc tgagaagcgc
     1801 agagaagtct cgagctcaag cttcgatggc gaccaccgtc agcactcagc gcgggccggt
     1861 gtacatcggt gagctcccgc aggacttcct ccgcatcacg cccacacagc agcagcggca
     1921 ggtccagctg gacgcccagg cggcccagca gctgcagtac ggaggcgcag tgggcaccgt
     1981 gggccgactg aacatcacgg tggtacaggc aaagttggcc aagaattacg gcatgacccg
     2041 catggacccc tactgccgac tgcgcctggg ctacgcggtg tacgagacgc ccacggcaca
     2101 caatggcgcc aagaatcccc gctggaataa ggtcatccac tgcacggtgc ccccaggcgt
     2161 ggactctttc tatctcgaga tcttcgatga gagagccttc tccatggacg accgcattgc
     2221 ctggacccac atcaccatcc cagagtccct gaggcagggc aaggtggagg acaagtggta
     2281 cagcctgagc gggaggcagg gggacgacaa ggagggcatg atcaacctcg tcatgtccta
     2341 cgcgctgctt ccagctgcca tggtgatgcc accccagccc gtggtcctga tgccaacagt
     2401 gtaccagcag ggcgttggct atgtgcccat cacagggatg cccgctgtct gtagccccgg
     2461 catggtgccc gtggccctgc ccccggccgc cgtgaacgcc cagccccgct gtagcgagga
     2521 ggacctgaaa gccatccagg acgcggcccc caacatggac caggaggtga tccgctccgt
     2581 gctggaagcc cagcgaggga acaaggatgc cgccatcaac tccctgctgc agatggggga
     2641 ggagccatag gaattcggat ccgcggccgc atagataact gatccagtgt gctggaatta
     2701 attcgctgtc tgcgagggcc agctgttggg gtgagtactc cctctcaaaa gcgggcatga
     2761 cttctgcgct aagattgtca gtttccaaaa acgaggagga tttgatattc acctggcccg
     2821 cggtgatgcc tttgagggtg gccgcgtcca tctggtcaga aaagacaatc tttttgttgt
     2881 caagcttgag gtgtggcagg cttgagatct ggccatacac ttgagtgaca atgacatcca
     2941 ctttgccttt ctctccacag gtgtccactc ccaggtccaa ctgcaggtcg agcatgcatc
     3001 tagggcggcc aattccgccc ctctcccccc cccccctctc cctccccccc ccctaacgtt
     3061 actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc
     3121 atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc
     3181 attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag
     3241 gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg
     3301 cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat
     3361 acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga
     3421 gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc
     3481 cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg
     3541 ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacacga
     3601 tgataagctt gccacaaccc gggataattc ctgcagccaa tatgggatcg gccattgaac
     3661 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     3721 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     3781 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     3841 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     3901 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     3961 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     4021 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     4081 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     4141 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgatgatc
     4201 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     4261 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     4321 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     4381 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     4441 tctgagggga tcaattctct agagctcgct gatcagcctc gactgtgcct tctagttgcc
     4501 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     4561 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     4621 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     4681 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcga
     4741 gtgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     4801 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     4861 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     4921 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4981 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5041 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5101 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5161 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5221 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5281 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5341 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5401 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5461 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5521 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5581 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5641 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     5701 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5761 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5821 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     5881 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     5941 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6001 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6061 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6121 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6181 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6241 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6301 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6361 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6421 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6481 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6541 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6601 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6661 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     6721 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     6781 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     6841 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     6901 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     6961 acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat
     7021 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg
     7081 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaa
//
