LOCUS       sequence-350117-        7130 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  myc-BioID fused to human Tollip 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 7130)
  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 7130)
  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
FEATURES             Location/Qualifiers
     source          1..7130
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        43..422
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        423..626
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     577..597
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     671..690
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        671..689
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             769..798
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             join(802..1149,1150..1152,1153..1761)
                     /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          2685..2914
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2897..2916
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    3092..3554
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3148..3165)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3375..3394
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3590..4393
                     /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(3653..3672)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4263..4282
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4425..4442)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4431..4655
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4702..4721
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4781..4797)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4781..4797)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4794..4816)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4805..4821
                     /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(4829..4835,4836..4853,4854..4859))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4874..4895
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5012..5029)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5183..5771)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5263..5282)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5942..6733,6734..6802))
                     /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     6565..6584
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6803..6907)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6982..7001)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
       61 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
      121 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      181 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      241 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      301 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      361 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      421 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
      481 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      541 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
      601 ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact gcttactggc
      661 ttatcgaaat taatacgact cactataggg agacccaagc ttggtaccga gctcggatcg
      721 atatctgcgg cctagctagc gcttaaggcc tgttaaccgg tcaccatgga acaaaaactc
      781 atctcagaag aggatctcga caaggacaac accgtgcccc tgaagctgat cgccctgctg
      841 gccaacggcg agttccactc tggcgagcag ctgggagaga ccctgggaat gagcagagcc
      901 gccatcaaca agcacatcca gacactgaga gactggggag tggacgtgtt caccgtgcct
      961 ggcaagggct acagcctgcc tgagcctatc cagctgctga acgccaagca gatcctggga
     1021 cagctggatg gcggaagcgt ggccgtgctg cctgtgatcg actccaccaa tcagtacctg
     1081 ctggacagaa tcggagagct gaagtccggc gacgcctgca tcgccgagta ccagcaggct
     1141 ggcagaggag gcagaggacg gaagtggttc agcccattcg gagccaacct gtacctgtcc
     1201 atgttctgga gactggagca gggacctgct gctgccatcg gactgagtct ggtgatcgga
     1261 atcgtgatgg ccgaggtgct gagaaagctg ggagccgaca aggtgagagt gaagtggcct
     1321 aatgacctgt acctccagga ccgcaagctg gctggcatcc tggtggagct gacaggcaag
     1381 acaggcgatg ccgctcagat cgtgatcgga gccggaatca acatggccat gagaagagtg
     1441 gaggagagcg tggtgaacca gggctggatc accctgcagg aggctggcat caacctggac
     1501 cggaacaccc tggccgccat gctgatcaga gagctgagag ccgctctgga gctgttcgag
     1561 caggagggac tggctcctta cctgagcaga tgggagaagc tggacaactt catcaacaga
     1621 cctgtgaagc tgatcatcgg cgacaaggaa atcttcggca tctccagagg aatcgacaag
     1681 cagggagctc tgctgctgga gcaggacgga atcatcaagc cctggatggg cggagaaatc
     1741 tccctgagaa gcgcagagaa gtctcgagct caagcttcga tggcgaccac cgtcagcact
     1801 cagcgcgggc cggtgtacat cggtgagctc ccgcaggact tcctccgcat cacgcccaca
     1861 cagcagcagc ggcaggtcca gctggacgcc caggcggccc agcagctgca gtacggaggc
     1921 gcagtgggca ccgtgggccg actgaacatc acggtggtac aggcaaagtt ggccaagaat
     1981 tacggcatga cccgcatgga cccctactgc cgactgcgcc tgggctacgc ggtgtacgag
     2041 acgcccacgg cacacaatgg cgccaagaat ccccgctgga ataaggtcat ccactgcacg
     2101 gtgcccccag gcgtggactc tttctatctc gagatcttcg atgagagagc cttctccatg
     2161 gacgaccgca ttgcctggac ccacatcacc atcccagagt ccctgaggca gggcaaggtg
     2221 gaggacaagt ggtacagcct gagcgggagg cagggggacg acaaggaggg catgatcaac
     2281 ctcgtcatgt cctacgcgct gcttccagct gccatggtga tgccacccca gcccgtggtc
     2341 ctgatgccaa cagtgtacca gcagggcgtt ggctatgtgc ccatcacagg gatgcccgct
     2401 gtctgtagcc ccggcatggt gcccgtggcc ctgcccccgg ccgccgtgaa cgcccagccc
     2461 cgctgtagcg aggaggacct gaaagccatc caggacatgt tccccaacat ggaccaggag
     2521 gtgatccgct ccgtgctgga agcccagcga gggaacaagg atgccgccat caactccctg
     2581 ctgcagatgg gggaggagcc ataggaattc ggatccgcgg ccgcatagat aactgatcca
     2641 gtgtgctgga attaattcgc tgtctgcgag ggccagctgt tggggtgagt actccctctc
     2701 aaaagcgggc atgacttctg cgctaagatt gtcagtttcc aaaaacgagg aggatttgat
     2761 attcacctgg cccgcggtga tgcctttgag ggtggccgcg tccatctggt cagaaaagac
     2821 aatctttttg ttgtcaagct tgaggtgtgg caggcttgag atctggccat acacttgagt
     2881 gacaatgaca tccactttgc ctttctctcc acaggtgtcc actcccaggt ccaactgcag
     2941 gtcgagcatg catctagggc ggccaattcc gcccctctcc cccccccccc tccccccccc
     3001 ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc tatatgttat
     3061 tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc cctgtcttct
     3121 tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg
     3181 tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct gtagcgaccc
     3241 tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa aagccacgtg
     3301 tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt tggatagttg
     3361 tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag gatgcccaga
     3421 aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt acatgtgttt
     3481 agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa
     3541 aaacacgatg ataagcttgc cacaacccgg gataattcct gcagccaata tgggatcggc
     3601 cattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     3661 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     3721 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     3781 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     3841 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     3901 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     3961 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     4021 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     4081 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     4141 cgatgatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     4201 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     4261 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     4321 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     4381 cgagttcttc tgaggggatc aattctctag agctcgctga tcagcctcga ctgtgccttc
     4441 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     4501 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     4561 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     4621 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     4681 gggctcgagt gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     4741 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     4801 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     4861 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     4921 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4981 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     5041 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     5101 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     5161 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5221 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5281 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5341 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5401 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5461 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5521 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5581 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     5641 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5701 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5761 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5821 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5881 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5941 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     6001 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     6061 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     6121 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6181 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6241 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6301 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6361 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6421 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6481 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6541 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6601 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6661 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6721 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6781 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6841 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6901 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
     6961 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
     7021 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
     7081 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
//
