LOCUS       sequence-455730-        4384 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 4384)
  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 4384)
  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: pML531_TOMM20-C-term_XTEN80-mEGFP-3xHA_pTMPLT
FEATURES             Location/Qualifiers
     source          1..4384
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     99..121
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     113..130
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     114..130
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             949..972
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(988..990,991..1701)
                     /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(1030..1051)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1291..1310)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1638..1659
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1714..1794
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(2402..2418)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2402..2418)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2415..2437)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(2590..3178)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2670..2689)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(3295..3319)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(3419..4210,4211..4279))
                     /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     4042..4061
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4280..4384)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacatttccc cgaaaagtgc cagatacctg aaacaaaacc catcgtacgg ccaaggaagt
       61 ctccaataac tgtgatccac cacaagcgcc agggttttcc cagtcacgac gttgtaaaac
      121 gacggccagt catgcataat ccgcacgcat ctggaataag gaagtgccat tccgcctgac
      181 ctggagctta ctggtgtcac ttcggtaaaa ccctgtctgc accaaaaata aaaaaaaatt
      241 agccgggcat ggtggcatga gtctgtggtt ccagctactc aggaggctga ggtgggggag
      301 gatcgcttga gcccaggagt cagaggttgc agtgagctga gattgtgcta ctgcactcca
      361 gcctgggtta cagagacccc atctgaatta aaaacatata taatgtaatg atctgcctcc
      421 tttgttaact tgacttttga aatgggattg tcagtagtat gatcattgtt ttcttggatg
      481 ccgactgtgt gtaaagtgtt acattttgaa ttaaatgtca gaatgggtga actttactaa
      541 gattcaattc tttgaataca aagagcattt tattttgaag ttagaatcct aattaaatgc
      601 ttatgacact ttaaaaaatt attttttttt ttctttcaga gaattgtaag tgctcagagc
      661 ttggctgaag acgacgtcga atctggtggt acttccgggg gagggccgag ctctggcgca
      721 cccccaccaa gtggagggtc acctgccggg tccccaacat ctactgaaga aggcaccagc
      781 gaatccgcaa cgcccgagtc aggccctggt acctccacag aaccatctga aggtagtgcg
      841 cctggttccc cagctggaag ccctacttcc accgaagaag gcacgtcaac cgaaccaagt
      901 gaaggatctg cccctgggac cagcactgaa ccatctgagg gtggcggatt ggaagttttg
      961 tttcaaggtc caggatctaa cggcagcgtg agcaagggcg aggagctgtt caccggggtg
     1021 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     1081 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     1141 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     1201 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     1261 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     1321 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     1381 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     1441 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     1501 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     1561 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccaagctgag caaagacccc
     1621 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     1681 ggcatggacg agctgtacaa ggctggagct ggctacccat acgatgttcc agattacgct
     1741 tatccgtatg acgtcccgga ctatgcatac ccctacgatg tacccgatta cgccggtgca
     1801 gggagtggaa gttgagaaac aaatgtcaac ataataaaat ctcagttaaa aatattttaa
     1861 aaattcttgg tagttgagca gctctggggg aataagggca aatatgcttg ttatgaacta
     1921 cactgaaatc taccaaagtt aatgtttact ttgtgtagat ccatttgtct attttattta
     1981 tttttcccag tgaaaagtgt attttgatag agaacttttc attctataaa tacactatga
     2041 gttactaaaa tatcatggat tttgtttatt cctgaaacat agttacatag ttaaactgta
     2101 catatgacat ggcttatgtt aaaaataccc agtgctcagt tttgaaagat aggcaaaaaa
     2161 aaaaagtata ggagaaactg aagaatgtac acttttttag agggcacatt ttgctgtaaa
     2221 tctggaaatt tgatagactt gactgtgttt gtgaaaactg agcattaaag gttttgattg
     2281 atcctttctt tccatttaat ctctgagacg taaatatgtg aggtgcgtca gaagaccgtc
     2341 agtgaaggct aggtggaggc tcagtgatga taagtctgcg atggtggatg catgtgtcat
     2401 ggtcatagct gtttcctgtg tgaaattgtt atccgctcag agggcacaat cctattccgc
     2461 gctatccgac aatctccaag acattaggtg gagttcagtt cggcgtatgg catatgtcgc
     2521 tggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     2581 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     2641 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     2701 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     2761 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     2821 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     2881 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     2941 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     3001 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     3061 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     3121 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     3181 agatcctttg atcttttcta cggggtctga cgctctattc aacaaagccg ccgtcccgtc
     3241 aagtcagcgt aaatgggtag ggggcttcaa atcgtcctcg tgataccaat tcggagcctg
     3301 cttttttgta caaacttgtt gataatggca attcaaggat cttcacctag atccttttaa
     3361 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     3421 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     3481 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     3541 gtgctgcaat gataccgcga gagccacgct caccggctcc agatttatca gcaataaacc
     3601 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     3661 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3721 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     3781 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     3841 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     3901 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     3961 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4021 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4081 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4141 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4201 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4261 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4321 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     4381 cgcg
//
