LOCUS       40924_785        8694 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Soybean APEX2 anchored to cytosolic face of ER membrane with 
            N-terminal targeting sequence of residues 1-27 of ER-resident P450 
            oxidase 2C1..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8694)
  AUTHORS   Lam SS, Martell JD, Kamer KJ, Deerinck TJ, Ellisman MH, Mootha VK, 
            Ting AY
  TITLE     Directed evolution of APEX2 for electron microscopy and proximity 
            labeling.
  JOURNAL   Nat Methods. 2014 Nov 24. doi: 10.1038/nmeth.3179.
  PUBMED    25419960
REFERENCE   2  (bases 1 to 8694)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8694)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2014 Nov 24. doi: 10.1038/nmeth.3179."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8694
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        105..408
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        409..612
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     protein_bind    635..659
                     /label=attB1
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             836..1582
                     /codon_start=1
                     /label=APEX2
                     /note="soybean ascorbate peroxidase, engineered to serve as
                     a monomeric tag for electron microscopy and live-cell 
                     proteomics (Lam et al., 2015)"
                     /translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
                     FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
                     EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
                     IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
                     YAADEDAFFADYAEAHQKLSELGFADA"
     CDS             1583..1624
                     /codon_start=1
                     /label=V5 tag
                     /note="epitope tag from simian virus 5"
                     /translation="GKPIPNPLLGLDST"
     protein_bind    complement(1632..1656)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1658..1699
                     /codon_start=1
                     /label=V5 tag
                     /note="epitope tag from simian virus 5"
                     /translation="GKPIPNPLLGLDST"
     misc_feature    1741..2329
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     LTR             2401..2634
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2712..2846
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      2873..3008
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        complement(3029..3047)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3057..3073)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      3215..3670
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        3696..3800
                     /label=AmpR promoter
     CDS             3801..4658
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      4832..5420
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5574..5591
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5708..5729
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        5744..5774
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5782..5798
                     /label=lac operator
                     /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)."
     primer_bind     5806..5822
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5843..5861
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5889..6115
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6116..6296
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6343..6468
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     misc_feature    6961..7194
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7379..7423
                     /codon_start=1
                     /label=gp41 peptide
                     /note="antigenic peptide corresponding to amino acids 655
                     to 669 of the HIV envelope protein gp41 (Lutje Hulsik et 
                     al., 2013)"
                     /translation="KNEQELLELDKWASL"
     promoter        7598..8098
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     CDS             8110..8505
                     /codon_start=1
                     /label=BSD
                     /note="blasticidin S deaminase"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    8565..8682
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
ORIGIN
        1 agggacagca gagatccact ttggcgccgg ctcgaggggg cccggggtta ttaatagtaa
       61 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
      121 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
      181 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
      241 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
      301 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
      361 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
      421 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
      481 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
      541 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
      601 ataagcagag ctctctggct aactgtcggg atcaacaagt ttgtacaaaa aagcaggctt
      661 cgaaggagat agaaccatgg accctgtggt cgtgctgggg ctgtgcctga gctgtctgct
      721 gctgctgagc ctgtggaagc agtcctacgg aggcggggga tcaggaagcg gcggccgcgg
      781 atcaggaagc ggatcaggaa gcggatcagg aagcggatca ggaagcggat caggaggaaa
      841 gtcttaccca actgtgagtg ctgattacca ggacgccgtt gagaaggcga agaagaagct
      901 cagaggcttc atcgctgaga agagatgcgc tcctctaatg ctccgtttgg cattccactc
      961 tgctggaacc tttgacaagg gcacgaagac cggtggaccc ttcggaacca tcaagcaccc
     1021 tgccgaactg gctcacagcg ctaacaacgg tcttgacatc gctgttaggc ttttggagcc
     1081 actcaaggcg gagttcccta ttttgagcta cgccgatttc taccagttgg ctggcgttgt
     1141 tgccgttgag gtcacgggtg gacctaaggt tccattccac cctggaagag aggacaagcc
     1201 tgagccacca ccagagggtc gcttgcccga tcccactaag ggttctgacc atttgagaga
     1261 tgtgtttggc aaagctatgg ggcttactga ccaagatatc gttgctctat ctgggggtca
     1321 cactattgga gctgcacaca aggagcgttc tggatttgag ggtccctgga cctctaatcc
     1381 tcttattttc gacaactcat acttcacgga gttgttgagt ggtgagaagg aaggtctcct
     1441 tcagctacct tctgacaagg ctcttttgtc tgaccctgta ttccgccctc tcgttgacaa
     1501 atatgcagcg gacgaagatg ccttctttgc tgattacgct gaggctcacc aaaagctttc
     1561 cgagcttggg tttgctgatg ccggcaagcc catccccaac cccctgctgg gcctggacag
     1621 cacctgataa gacccagctt tcttgtacaa agtggttggt aagcctatcc ctaaccctct
     1681 cctcggtctc gattctacgt agtaatgagc tagcgctaac cggtggcgcg ttaagtcgac
     1741 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     1801 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     1861 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     1921 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     1981 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     2041 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     2101 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
     2161 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     2221 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     2281 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcg tcgactttaa
     2341 gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac
     2401 tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc
     2461 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     2521 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     2581 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtacgt
     2641 atagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag
     2701 agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     2761 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     2821 tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc ccatcccgcc
     2881 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     2941 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     3001 tggaggccta gggacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg
     3061 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     3121 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     3181 tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     3241 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3301 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3361 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3421 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     3481 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     3541 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     3601 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     3661 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     3721 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     3781 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     3841 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     3901 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     3961 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     4021 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     4081 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     4141 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     4201 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     4261 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     4321 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     4381 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     4441 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     4501 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     4561 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     4621 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     4681 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     4741 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4801 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4861 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4921 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     4981 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     5041 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     5101 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     5161 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     5221 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     5281 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     5341 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     5401 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     5461 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     5521 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     5581 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     5641 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     5701 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     5761 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     5821 accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctgc
     5881 aagcttaatg tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta
     5941 gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg
     6001 gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac
     6061 tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc
     6121 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
     6181 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
     6241 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
     6301 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
     6361 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
     6421 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
     6481 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
     6541 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
     6601 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
     6661 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
     6721 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
     6781 aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat
     6841 gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg
     6901 agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat
     6961 aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat
     7021 gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt
     7081 gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca
     7141 gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat
     7201 ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag
     7261 taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat
     7321 taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa
     7381 gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat
     7441 aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt
     7501 aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt
     7561 atcgtttcag acccacctcc caaccccgag gggacccttg cgccttttcc aaggcagccc
     7621 tgggtttgcg cagggacgcg gctgctctgg gcgtggttcc gggaaacgca gcggcgccga
     7681 ccctgggtct cgcacattct tcacgtccgt tcgcagcgtc acccggatct tcgccgctac
     7741 ccttgtgggc cccccggcga cgcttcctgc tccgccccta agtcgggaag gttccttgcg
     7801 gttcgcggcg tgccggacgt gacaaacgga agccgcacgt ctcactagta ccctcgcaga
     7861 cggacagcgc cagggagcaa tggcagcgcg ccgaccgcga tgggctgtgg ccaatagcgg
     7921 ctgctcagca gggcgcgccg agagcagcgg ccgggaaggg gcggtgcggg aggcggggtg
     7981 tggggcggta gtgtgggccc tgttcctgcc cgcgcggtgt tccgcattct gcaagcctcc
     8041 ggagcgcacg tcggcagtcg gctccctcgt tgaccgaatc accgacctct ctccccaggg
     8101 ggtaccacca tggccaagcc tttgtctcaa gaagaatcca ccctcattga aagagcaacg
     8161 gctacaatca acagcatccc catctctgaa gactacagcg tcgccagcgc agctctctct
     8221 agcgacggcc gcatcttcac tggtgtcaat gtatatcatt ttactggggg accttgtgca
     8281 gaactcgtgg tgctgggcac tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc
     8341 gcgatcggaa atgagaacag gggcatcttg agcccctgcg gacggtgccg acaggtgctt
     8401 ctcgatctgc atcctgggat caaagccata gtgaaggaca gtgatggaca gccgacggca
     8461 gttgggattc gtgaattgct gccctctggt tatgtgtggg agggcctgca gctgcagtag
     8521 taagaattct agatcttgag acaaatggca gtattcatcc acaattttaa aagaaaaggg
     8581 gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa
     8641 actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttac
//
