LOCUS       sequence-453976-        3993 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology repair template for genome engineering. Organelle 
            endogenous marker expressing GFP and epitope tags for mass 
            spectrometry..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3993)
  AUTHORS   Hein MY, Peng D, Todorova V, McCarthy F, Kim K, Liu C, Savy L, 
            Januel C, Baltazar-Nunez R, Sekhar M, Vaid S, Bax S, Vangipuram M, 
            Burgess J, Njoya L, Wang E, Ivanov IE, Byrum JR, Pradeep S, Gonzalez
            CG, Aniseia Y, Creery JS, McMorrow AH, Sunshine S, Yeung-Levy S, 
            DeFelice BC, Mehta SB, Itzhak DN, Elias JE, Leonetti MD
  TITLE     Global organelle profiling reveals subcellular localization and 
            remodeling at proteome scale.
  JOURNAL   Cell. 2024 Dec 26:S0092-8674(24)01344-8. doi: 
            10.1016/j.cell.2024.11.028.
  PUBMED    39742809
REFERENCE   2  (bases 1 to 3993)
  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: "Cell. 2024 
            Dec 26:S0092-8674(24)01344-8. doi: 10.1016/j.cell.2024.11.028."
COMMENT     Sequence Label: pML364_PSMB7-C-term_XTEN80-mEGFP-3xHA_pTMPLT
FEATURES             Location/Qualifiers
     source          1..3993
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     137..156
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(375..479)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     578..600
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     592..609
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     593..609
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1250..1273
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(1289..1291,1292..2002)
                     /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(1331..1352)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1592..1611)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1939..1960
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2015..2095
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(2490..2506)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2490..2506)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2503..2525)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(2678..3266)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2758..2777)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(3383..3407)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(3507..3993,1..305,306..374))
                     /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"
ORIGIN
        1 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
       61 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
      121 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
      181 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
      241 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
      301 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
      361 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
      421 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
      481 acatttcccc gaaaagtgcc agatacctga aacaaaaccc atcgtacggc caaggaagtc
      541 tccaataact gtgatccacc acaagcgcca gggttttccc agtcacgacg ttgtaaaacg
      601 acggccagtc atgcataatc cgcacgcatc tggaataagg aagtgccatt ccgcctgacc
      661 tggagcttac tggtgtcact tcgcaccccc attttgcagc acttgagaac tgaggaagtt
      721 gaatccccca agatcacaga gctgggatcg ggagagccaa gattccagtc cagcctgctc
      781 actgcgttgc ctgaatattt attttcactg tcttctgccc tctggcacct tgaggagaat
      841 cctaactctg tgcttttgtt tttctcttac taggcttggc cggtacaggt gtgagaaagg
      901 gactactgca gtcctcactg agaaaatcac tcctctggag attgaggtgc tggaagaaac
      961 agtccaaaca atggacactt ccagtggtgg tacttccggg ggagggccga gctctggcgc
     1021 acccccacca agtggagggt cacctgccgg gtccccaaca tctactgaag aaggcaccag
     1081 cgaatccgca acgcccgagt caggccctgg tacctccaca gaaccatctg aaggtagtgc
     1141 gcctggttcc ccagctggaa gccctacttc caccgaagaa ggcacgtcaa ccgaaccaag
     1201 tgaaggatct gcccctggga ccagcactga accatctgag ggtggcggat tggaagtttt
     1261 gtttcaaggt ccaggatcta acggcagcgt gagcaagggc gaggagctgt tcaccggggt
     1321 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     1381 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     1441 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     1501 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     1561 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     1621 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     1681 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     1741 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     1801 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     1861 ccccgtgctg ctgcccgaca accactacct gagcacccag tccaagctga gcaaagaccc
     1921 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     1981 cggcatggac gagctgtaca aggctggagc tggctaccca tacgatgttc cagattacgc
     2041 ttatccgtat gacgtcccgg actatgcata cccctacgat gtacccgatt acgccggtgc
     2101 agggagtgga agttgaatgg catcagtggg tggctggccg cggttctgga aggtggtgag
     2161 cattgaggcc cagtaagaca ctcatgtggc tagtgtttgc cgaatgaaac tcaactcaat
     2221 aaaaaacaaa aaccaaattg ggcagctgag gtctgggcac tgtgtggtgt ttttgggggc
     2281 agtagggggc tgctaaggcc attggcctgg cccttcaggg tgctgatcct ccttgagcac
     2341 tggcctaggc tcagaggaga gcgtaggagc catgcgcccc tcctgaggac agataggttc
     2401 tgtctgctgt cagcgtcaga agaccgtcag tgaaggctag gtggaggctc agtgatgata
     2461 agtctgcgat ggtggatgca tgtgtcatgg tcatagctgt ttcctgtgtg aaattgttat
     2521 ccgctcagag ggcacaatcc tattccgcgc tatccgacaa tctccaagac attaggtgga
     2581 gttcagttcg gcgtatggca tatgtcgctg gaaagaacat gtgagcaaaa ggccagcaaa
     2641 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     2701 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     2761 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     2821 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     2881 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     2941 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     3001 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     3061 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     3121 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     3181 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     3241 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     3301 ctctattcaa caaagccgcc gtcccgtcaa gtcagcgtaa atgggtaggg ggcttcaaat
     3361 cgtcctcgtg ataccaattc ggagcctgct tttttgtaca aacttgttga taatggcaat
     3421 tcaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     3481 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     3541 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     3601 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga gccacgctca
     3661 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     3721 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     3781 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     3841 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     3901 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     3961 agtaagttgg ccgcagtgtt atcactcatg gtt
//
