LOCUS       sequence-457382-        4479 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 4479)
  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 4479)
  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: pML460_MAP1LC3B-N-term_XTEN80-mEGFP-3xHA_pTMPLT
FEATURES             Location/Qualifiers
     source          1..4479
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     60..79
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(298..402)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     516..532
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     621..637
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1251..1331
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             join(1344..1346,1347..2057)
                     /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(1386..1407)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1647..1666)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1994..2015
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2067..2090
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2969..2985)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2969..2985)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2982..3004)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(3157..3745)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3237..3256)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(3792..3816)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(3916..4479,1..228,229..297))
                     /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 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
       61 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
      121 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
      181 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
      241 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
      301 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
      361 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
      421 gccagatacc tgaaacaaaa cccatcgtac ggccaaggaa gtctccaata actgtgatcc
      481 accacaagcg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtcatgcata
      541 atccgcacgc atctggaata aggaagtctc caataactgt gatccaccac aagcgccagg
      601 gttttcccag tcacgacgtt gtaaaacgac ggccagtcat gcataatccg cacgcatctg
      661 gaataaggaa gtgccattcc gcctgacctg gagcttactg gtgtcacttc aggatggtcg
      721 gatttgcccc atgtcccttc ctctgacccc tccctcaaga gtgccccggg acaccccgcc
      781 tgtggctcag cctcccccgc cccgcgctgc catctcctca gggccgggca gcaggctccc
      841 gagcgcccac agacccgggg tgcggcccag cccacaaccg tcacctcagg ggcctcaggc
      901 gcccagcggt gctgggcggg gctggggcac gaccgggagc atgcgcagag cgcgcgtttc
      961 gcccatcgcg cacgcgcaca cacctgctcc gcccccacgc tgcgtgccgc tgctgggttc
     1021 cgccacgccc gtcatggcgg cggccccggc cggctctggc cccgcccctc ggtgacgcgt
     1081 cgcgagtcac ctgaccaggc tgcgggctga ggagatacaa gggaagtggc tatcgccaga
     1141 gtcggattcg ccgccgcagc agccgccgcc cccgggagcc gccgggaccc tcgcgtcgtc
     1201 gccgccgccg ccgcccagat ccctgcacca tgtctggtgg tacttccggg tatccatacg
     1261 atgttccaga ttacgcttat ccgtatgacg tcccggacta tgcatacccc tatgatgtac
     1321 ccgactacgc cgctggagct ggcgtgagca agggcgagga gctgttcacc ggggtggtgc
     1381 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     1441 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     1501 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     1561 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     1621 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     1681 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     1741 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     1801 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     1861 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     1921 tgctgctgcc cgacaaccac tacctgagca cccagtccaa gctgagcaaa gaccccaacg
     1981 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     2041 tggacgagct gtacaagggt ggcggattgg aagttttgtt tcaaggtcca ggatctaacg
     2101 gcagcggagg gccgagctct ggcgcacccc caccaagtgg agggtcacct gccgggtccc
     2161 caacatctac tgaagaaggc accagcgaat ccgcaacgcc cgagtcaggc cctggtacct
     2221 ccacagaacc atctgaaggt agtgcgcctg gttccccagc tggaagccct acttccaccg
     2281 aagaaggcac gtcaaccgaa ccaagtgaag gatctgcccc tgggaccagc actgaaccat
     2341 ctgagggtgc agggagtgga agtccgtcgg agaagacctt caagcagcgc cgcaccttcg
     2401 gtgagtgtcg ccgcgagggc ggcgggtgcg gcggggccgg ggtccgagct gtggagggcg
     2461 gcagggcctg ggacgccgtg aggggtcggg gccgagccgg gaggccgagc ggggccggtg
     2521 gctgccggcc ggcggggccg agggatgcgg ggcccggggc cccgtgaggg acccaggccg
     2581 ggaccgagcc gggagggcca ggggctggtc tgggccgggg cggcctgcgg acctctcgga
     2641 ggcctgggag gaggcagccg gcggagggcg ggcgggggcc ggtgcgctcg gggccccggg
     2701 cctgctgaat caccccgcgc cctccgcggc ggggtgcctg ccgtaccccg gccaccgccg
     2761 cccccgcaag cgcgctgcgg gcgagggtcg cgcttctggg gcccaacagc ccccggggcc
     2821 gcgctgggcc gttcggctct gaagcggggc tgcgccggga cccagcgcgt caccttcagg
     2881 gctggttttc cccgtcagaa gaccgtcagt gaaggctagg tggaggctca gtgatgataa
     2941 gtctgcgatg gtggatgcat gtgtcatggt catagctgtt tcctgtgtga aattgttatc
     3001 cgctcagagg gcacaatcct attccgcgct atccgacaat ctccaagaca ttaggtggag
     3061 ttcagttcgg cgtatggcat atgtcgctgg aaagaacatg tgagcaaaag gccagcaaaa
     3121 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     3181 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     3241 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     3301 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     3361 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     3421 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     3481 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     3541 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     3601 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     3661 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     3721 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     3781 tctattcaac aaascygctt ttttgtacaa acttgttgat aatggcaatt caaggatctt
     3841 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     3901 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     3961 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     4021 cttaccatct ggccccagtg ctgcaatgat accgcgagag ccacgctcac cggctccaga
     4081 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     4141 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     4201 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     4261 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     4321 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     4381 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     4441 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaa
//
