LOCUS       sequence-455731-        3976 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 3976)
  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 3976)
  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: pML530_EEA1-N-term_XTEN80-mEGFP-3xHA_pTMPLT
FEATURES             Location/Qualifiers
     source          1..3976
                     /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             443..523
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             join(536..538,539..1249)
                     /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(578..599)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(839..858)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1186..1207
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1259..1282
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1994..2010)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1994..2010)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2007..2029)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(2182..2770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2262..2281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(2887..2911)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(3011..3802,3803..3871))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASQQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3634..3653
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3872..3976)
                     /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 ttccaggctg cctagtcgtt cgctcgctct ctctcgcgtg
      241 ctccctctcg ccgcctgtgg ggaagtcggg gccgcccgag cggccgtcgc caccgcctcg
      301 ccccgacctc cttcccggtc ccctcccggc ccagtccctc ccgcccgcgc accccgagta
      361 gtgagtggcc ccgcgcaggg tctggagagt caccgcggcg gcgccgggtg gtggttaaac
      421 catgtctggt ggtacttccg ggtacccata cgatgttcca gattacgctt atccgtatga
      481 cgtcccggac tatgcatacc cctacgatgt acccgattac gccgctggag ctggcgtgag
      541 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
      601 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
      661 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
      721 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
      781 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
      841 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
      901 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
      961 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     1021 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     1081 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     1141 cacccagtcc aagctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     1201 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg gtggcggatt
     1261 ggaagttttg tttcaaggtc caggatctaa cggcagcgga gggccgagct ctggcgcacc
     1321 cccaccaagt ggagggtcac ctgccgggtc cccaacatct actgaagaag gcaccagcga
     1381 atccgcaacg cccgagtcag gccctggtac ctccacagaa ccatctgaag gtagtgcgcc
     1441 tggttcccca gctggaagcc ctacttccac cgaagaaggc acgtcaaccg aaccaagtga
     1501 aggatctgcc cctgggacca gcactgaacc atctgagggt gcagggagtg gaagtttaag
     1561 gaggatttta cagagggtaa gagagtgaaa ccgtctttct ggcagcacgt gagcgggaca
     1621 gccggagctc ggggccgcgg cgcgggcgcc cctcctcagc gcgggctcct tccttcccta
     1681 ggccccacag cccggcaggc gcttaggcct cggtcgcccg gcccgggggc ggggagggcg
     1741 ggtggggccg gcgcggcggg gcggcaggca gagccctggt ggccccgcgc gtccaggcct
     1801 ggcgcccgct ccggcgcggg gctccgtgtg tgcgagcggg ctgcgagctg gagggtgtgg
     1861 tgccggcgcg ttcctcccgg gaaccctgag tgggagtgaa aatggagagt tggtgcccgt
     1921 cagaagaccg tcagtgaagg ctaggtggag gctcagtgat gataagtctg cgatggtgga
     1981 tgcatgtgtc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc agagggcaca
     2041 atcctattcc gcgctatccg acaatctcca agacattagg tggagttcag ttcggcgtat
     2101 ggcatatgtc gctggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     2161 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     2221 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     2281 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     2341 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     2401 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     2461 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     2521 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     2581 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     2641 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     2701 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     2761 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctctat tcaacaaagc
     2821 cgccgtcccg tcaagtcagc gtaaatgggt agggggcttc aaatcgtcct cgtgatacca
     2881 attcggagcc tgcttttttg tacaaacttg ttgataatgg caattcaagg atcttcacct
     2941 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     3001 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     3061 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3121 catctggccc cagtgctgca atgataccgc gagagccacg ctcaccggct ccagatttat
     3181 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     3241 cctccatcca gtctattaat tgttgctggg aagctagagt aagtagttcg ccagttaata
     3301 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     3361 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     3421 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     3481 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     3541 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     3601 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     3661 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     3721 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     3781 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     3841 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     3901 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     3961 aaataggggt tccgcg
//
