LOCUS       sequence-421670-        6698 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses lipid-raft targeted APEX2 for proximity proteomics. 
            Vector: pcDNA(TM)4/TO. Insert: lipid raft target sequence (MGCVCSS) 
            + mCherry +V5 (GKPIPNPLLGLDST) +APEX2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6698)
  TITLE     B cell receptor-induced protein dynamics and the emerging role of 
            SUMOylation revealed by proximity proteomics
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6698)
  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: 
            "Unpublished"
COMMENT     Sequence Label: raft-APEX2
FEATURES             Location/Qualifiers
     source          1..6698
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(94..111)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    100..324
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      370..798
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(457..476)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     667..688
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(807..827)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        812..1141
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      992..1127
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1054..1073
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        1189..1236
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             1255..1629
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    1759..1880
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1796..1815)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1850..1869
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1929..1945)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1929..1945)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1942..1964)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1953..1969
                     /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(1977..1983,1984..2001,2002..2007))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2022..2043
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2160..2177)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2331..2919)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2411..2430)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3090..3881,3882..3950))
                     /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     3713..3732
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3951..4055)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4130..4149)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4321..4700
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4701..4904
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4855..4875
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    4906..4924
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4927..4945
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     4955..4979
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             5139..5843
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(5281..5299)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5549..5568
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             5904..5945
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             5946..6692
                     /codon_start=1
                     /product="soybean ascorbate peroxidase, engineered to serve
                     as a monomeric tag for electron microscopy and live-cell 
                     proteomics (Lam et al., 2015)"
                     /label=APEX2
                     /note="more active than the original APEX due to the A134P 
                     mutation"
                     /translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
                     FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
                     EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
                     IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
                     YAADEDAFFADYAEAHQKLSELGFADA"
ORIGIN
        1 ggatccacta gtccagtgtg gtggaattct gcagatatcc agcacagtgg cggccgctcg
       61 agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc
      121 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
      181 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
      241 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
      301 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg
      361 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
      421 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
      481 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
      541 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
      601 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
      661 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
      721 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
      781 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
      841 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
      901 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
      961 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     1021 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     1081 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     1141 aaagctcccg ggagcttgta tatccatttt cggatctgat cagcacgtgt tgacaattaa
     1201 tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta aaccatggcc
     1261 aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc
     1321 tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc
     1381 cgggacgacg tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc
     1441 ctggcctggg tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg
     1501 tccacgaact tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg
     1561 gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag
     1621 caggactgac acgtgctacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     1681 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     1741 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     1801 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     1861 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     1921 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     1981 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     2041 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     2101 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     2161 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     2221 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     2281 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     2341 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     2401 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     2461 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     2521 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     2581 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     2641 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     2701 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     2761 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     2821 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     2881 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     2941 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     3001 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     3061 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     3121 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     3181 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     3241 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     3301 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     3361 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     3421 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     3481 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     3541 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     3601 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     3661 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     3721 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     3781 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     3841 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     3901 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     3961 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     4021 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     4081 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
     4141 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     4201 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
     4261 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
     4321 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
     4381 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     4441 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
     4501 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
     4561 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
     4621 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
     4681 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
     4741 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
     4801 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
     4861 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata
     4921 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
     4981 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
     5041 tccggactct agcgtttaaa cttaagcttg gtaccgagct cggatccccg ctagccatgg
     5101 gctgtgtctg cagctcaaac cctgaaaaca acaacaacat ggtgagcaag ggcgaggata
     5161 acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc tccgtgaacg
     5221 gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg
     5281 ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc
     5341 agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga
     5401 agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg
     5461 tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc
     5521 tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg
     5581 aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagcaga
     5641 ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc tacaaggcca
     5701 agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac atcacctccc
     5761 acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc cactccaccg
     5821 gcggcatgga cgagctgtac aagtccggac tcagatctcg agctcaagct tcgaattctg
     5881 cagtcgacgg taccgcggat ccaggcaagc ccatccccaa ccccctgctg ggcctggaca
     5941 gcaccggaaa gtcttaccca actgtgagtg ctgattacca ggacgccgtt gagaaggcga
     6001 agaagaagct cagaggcttc atcgctgaga agagatgcgc tcctctaatg ctccgtttgg
     6061 cattccactc tgctggaacc tttgacaagg gcacgaagac cggtggaccc ttcggaacca
     6121 tcaagcaccc tgccgaactg gctcacagcg ctaacaacgg tcttgacatc gctgttaggc
     6181 ttttggagcc actcaaggcg gagttcccta ttttgagcta cgccgatttc taccagttgg
     6241 ctggcgttgt tgccgttgag gtcacgggtg gacctaaggt tccattccac cctggaagag
     6301 aggacaagcc tgagccacca ccagagggtc gcttgcccga tcccactaag ggttctgacc
     6361 atttgagaga tgtgtttggc aaagctatgg ggcttactga ccaagatatc gttgctctat
     6421 ctgggggtca cactattgga gctgcacaca aggagcgttc tggatttgag ggtccctgga
     6481 cctctaatcc tcttattttc gacaactcat acttcacgga gttgttgagt ggtgagaagg
     6541 aaggtctcct tcagctacct tctgacaagg ctcttttgtc tgaccctgta ttccgccctc
     6601 tcgttgataa atatgcagcg gacgaagatg ccttctttgc tgattacgct gaggctcacc
     6661 aaaagctttc cgagcttggg tttgctgatg cctaatga
//
