LOCUS       Exported                9076 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  HA-PEROXO Lentiviral Construct. Expresses a Peroxisomal Tag..
ACCESSION   .
VERSION     .
KEYWORDS    pLJC5-3XHA-EGFP-PEX26
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9076)
  AUTHORS   Ray GJ, Boydston EA, Shortt E, Wyant GA, Lourido S, Chen WW, 
            Sabatini DM
  TITLE     A PEROXO-Tag enables rapid isolation of peroxisomes from human cells
  JOURNAL   iScience, ISSN: 2589-0042, Vol: 0, Issue: 0, Page: 101109
REFERENCE   2  (bases 1 to 9076)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "iScience, 
            ISSN: 2589-0042, Vol: 0, Issue: 0, Page: 101109"
FEATURES             Location/Qualifiers
     source          1..9076
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..227
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             228..408
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     misc_feature    455..580
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1073..1306
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1491..1535
                     /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        1824..3035
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     2872..2891
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             3073..3099
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3112..3138
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3151..3177
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3190..3903
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3232..3253)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3493..3512)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3840..3861
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    4170..4287
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4336..4846
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4765..4783
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4868..5467
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4868..4887)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5364..5384
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5595..5828
                     /label=3 LTR (Delta-U3)
                     /note="self-inactivating 3 long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5900..6021
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5937..5956)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5991..6010
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6061..6196
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6123..6142
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(6216..6235)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6217..6235)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6245..6262)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6245..6261)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6254..6276)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6403..6858
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6490..6509)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6700..6721
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6885..6988
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6989..7849
                     /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(7207..7226)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8020..8608
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8509..8528
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8762..8779
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8896..8917
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        8932..8962
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8970..8986
                     /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     8975..8997
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8994..9010
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8994..9010
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     9029..9049
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        9031..9049
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"