LOCUS       sequence-453969-        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: pML600_COG8-C-term_XTEN80-mEGFP-3xHA_pTMPLT
FEATURES             Location/Qualifiers
     source          1..3993
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(57..73)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(57..73)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(70..92)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(245..833)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(325..344)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(950..974)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(1074..1865,1866..1934))
                     /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     1697..1716
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1935..2039)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2138..2160
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2152..2169
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2153..2169
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2810..2833
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(2849..2851,2852..3562)
                     /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(2891..2912)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3152..3171)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3499..3520
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3575..3655
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     regulatory      3939..3948
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 aggctaggtg gaggctcagt gatgataagt ctgcgatggt ggatgcatgt gtcatggtca
       61 tagctgtttc ctgtgtgaaa ttgttatccg ctcagagggc acaatcctat tccgcgctat
      121 ccgacaatct ccaagacatt aggtggagtt cagttcggcg tatggcatat gtcgctggaa
      181 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
      241 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
      301 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
      361 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
      421 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
      481 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
      541 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
      601 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
      661 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
      721 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
      781 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
      841 ctttgatctt ttctacgggg tctgacgctc tattcaacaa agccgccgtc ccgtcaagtc
      901 agcgtaaatg ggtagggggc ttcaaatcgt cctcgtgata ccaattcgga gcctgctttt
      961 ttgtacaaac ttgttgataa tggcaattca aggatcttca cctagatcct tttaaattaa
     1021 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     1081 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     1141 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     1201 gcaatgatac cgcgagagcc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     1261 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     1321 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     1381 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     1441 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     1501 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     1561 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     1621 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     1681 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     1741 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     1801 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     1861 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     1921 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     1981 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     2041 acatttcccc gaaaagtgcc agatacctga aacaaaaccc atcgtacggc caaggaagtc
     2101 tccaataact gtgatccacc acaagcgcca gggttttccc agtcacgacg ttgtaaaacg
     2161 acggccagtc atgcataatc cgcacgcatc tggaataagg aagtgccatt ccgcctgacc
     2221 tggagcttac tggtgtcact tcttattagt aactgttttc cattttgctt tccccacgtc
     2281 tgggttaggc attcctccca ctcagctctc caagtacggt aacctagggc atgtgaacat
     2341 cggcgccatt caggagcccc tcgcctttat cctgccaaag agagagacgc ttttcaccct
     2401 ggatgaccag gcgctggggc ccgagctcac agctccagca ccagagcctc ccgccgagga
     2461 gccacgcctg gagcccgcgg gcccagcctg cccggaggga gggcgagcgg agacgcaggc
     2521 cgaaccgccc agcgtggggc cctctggtgg tacttccggg ggagggccga gctctggcgc
     2581 acccccacca agtggagggt cacctgccgg gtccccaaca tctactgaag aaggcaccag
     2641 cgaatccgca acgcccgagt caggccctgg tacctccaca gaaccatctg aaggtagtgc
     2701 gcctggttcc ccagctggaa gccctacttc caccgaagaa ggcacgtcaa ccgaaccaag
     2761 tgaaggatct gcccctggga ccagcactga accatctgag ggtggcggat tggaagtttt
     2821 gtttcaaggt ccaggatcta acggcagcgt gagcaagggc gaggagctgt tcaccggggt
     2881 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     2941 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     3001 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     3061 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     3121 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     3181 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     3241 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     3301 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     3361 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     3421 ccccgtgctg ctgcccgaca accactacct gagcacccag tccaagctga gcaaagaccc
     3481 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     3541 cggcatggac gagctgtaca aggctggagc tggctatcca tacgatgttc cagattacgc
     3601 ttatccgtat gacgtcccgg actatgcata cccctatgat gtacccgact acgccggtgc
     3661 agggagtgga agttagccgg cgtcccctgc ctccagaacg cggtgcgtga ggttcccggc
     3721 gcggggtggc caggactgcc tcgccctgga aggtccgggg cgggttcggg aggcgggaag
     3781 gcgctcccgg gaggagagcg gaagggggcg gtgctcactg ccagtctaag caggaaggcg
     3841 gggatccagt cgcggcttcg ctgtgtgctg gccgacggcg cttcccggcg tgccccgcgc
     3901 tgcggccgga aggggcgggc cgggtcactg ttaagggggc cgccatggcc cggctgtggg
     3961 gcgcgctgag tctcgtcaga agaccgtcag tga
//
