LOCUS       Exported                8694 bp ds-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    ERM-APEX2
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)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2014 Nov 24. doi: 10.1038/nmeth.3179."
FEATURES             Location/Qualifiers
     source          1..8694
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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"
     primer_bind     563..583
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    635..659
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             836..1582
                     /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"
     CDS             1583..1624
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    complement(1632..1656)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1658..1699
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     misc_feature    1741..2329
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1794..1814)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2212..2223)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2401..2634
                     /label=3 LTR (Delta-U3)
                     /note="self-inactivating 3 long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2712..2833
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2749..2768)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2803..2822
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2873..3008
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2935..2954
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(3028..3047)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3029..3047)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3057..3074)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3057..3073)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3066..3088)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3215..3670
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3302..3321)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3512..3533
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3696..3800
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3801..4661
                     /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     complement(4019..4038)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4832..5420
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5321..5340
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5574..5591
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5708..5729
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /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
                     /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)."
     primer_bind     5787..5809
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5806..5822
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5806..5822
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5841..5861
                     /label=T3
                     /note="T3 promoter, forward primer"
     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
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        7598..8098
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     8017..8035
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             8110..8505
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    8565..8682
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"