LOCUS       sequence-452479-        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: pML333_LAMP1-C-term_XTEN80-mEGFP-3xHA_pTMPLT
FEATURES             Location/Qualifiers
     source          1..3993
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(124..228)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     327..349
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     341..358
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     342..358
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             999..1022
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(1038..1040,1041..1751)
                     /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(1080..1101)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1341..1360)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1688..1709
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1764..1844
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(2239..2255)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2239..2255)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2252..2274)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(2427..3015)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2507..2526)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(3132..3156)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(3256..3993,1..54,55..123))
                     /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     3879..3898
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
       61 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
      121 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
      181 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
      241 aaaagtgcca gatacctgaa acaaaaccca tcgtacggcc aaggaagtct ccaataactg
      301 tgatccacca caagcgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtca
      361 tgcataatcc gcacgcatct ggaataagga agtgccattc cgcctgacct ggagcttact
      421 ggtgtcactt ctctctgcag cggcttcccc aagtgacaat tccagctaga gctggaacct
      481 tccgccaggg ttcctctttg ccttttggct gatgtggggg agtggtgggg agagaggcct
      541 gtttgcaggc ccctgtgtga gcagagccct gacaccatcc gtctgtcttg gcagtggagg
      601 agtgtctgct ggacgagaac agcatgctga tccccatcgc tgtgggtggt gccctggcgg
      661 ggctggtcct catcgtcctc atcgcctacc tcgtcggcag gaagaggagt cacgcaggct
      721 accagactat cagtggtggt acttccgggg gagggccgag ctctggcgca cccccaccaa
      781 gtggagggtc acctgccggg tccccaacat ctactgaaga aggcaccagc gaatccgcaa
      841 cgcccgagtc aggccctggt acctccacag aaccatctga aggtagtgcg cctggttccc
      901 cagctggaag ccctacttcc accgaagaag gcacgtcaac cgaaccaagt gaaggatctg
      961 cccctgggac cagcactgaa ccatctgagg gtggcggatt ggaagttttg tttcaaggtc
     1021 caggatctaa cggcagcgtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     1081 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     1141 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     1201 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     1261 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     1321 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     1381 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     1441 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     1501 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     1561 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     1621 tgcccgacaa ccactacctg agcacccagt ccaagctgag caaagacccc aacgagaagc
     1681 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     1741 agctgtacaa ggctggagct ggctacccat acgatgttcc agattacgct tatccgtatg
     1801 acgtcccgga ctatgcatac ccctacgatg tacccgatta cgccggtgca gggagtggaa
     1861 gttagcctgg tgcacgcagg cacagcagct gcaggggcct ctgttccttt ctctgggctt
     1921 agggtcctgt cgaaggggag gcacactttc tggcaaacgt ttctcaaatc tgcttcatcc
     1981 aatgtgaagt tcatcttgca gcatttacta tgcacaacag agtaactatc gaaatgacgg
     2041 tgttaatttt gctaactggg ttaaatattt tgctaactgg ttaaacatta atatttacca
     2101 aagtaggatt ttgagggtgg gggtgctctc tctgaggggg tgggggtgcc gctgtctctg
     2161 agcgtcagaa gaccgtcagt gaaggctagg tggaggctca gtgatgataa gtctgcgatg
     2221 gtggatgcat gtgtcatggt catagctgtt tcctgtgtga aattgttatc cgctcagagg
     2281 gcacaatcct attccgcgct atccgacaat ctccaagaca ttaggtggag ttcagttcgg
     2341 cgtatggcat atgtcgctgg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     2401 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     2461 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     2521 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     2581 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     2641 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     2701 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     2761 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     2821 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     2881 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     2941 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     3001 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tctattcaac
     3061 aaagccgccg tcccgtcaag tcagcgtaaa tgggtagggg gcttcaaatc gtcctcgtga
     3121 taccaattcg gagcctgctt ttttgtacaa acttgttgat aatggcaatt caaggatctt
     3181 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     3241 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     3301 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     3361 cttaccatct ggccccagtg ctgcaatgat accgcgagag ccacgctcac cggctccaga
     3421 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     3481 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     3541 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     3601 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     3661 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     3721 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     3781 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     3841 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     3901 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     3961 accgctgttg agatccagtt cgatgtaacc cac
//
