LOCUS       sequence-452495-        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: pML389_YWHAB-C-term_XTEN80-mEGFP-3xHA_pTMPLT
FEATURES             Location/Qualifiers
     source          1..3993
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(117..221)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     320..342
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     334..351
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     335..351
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             992..1015
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(1031..1033,1034..1744)
                     /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(1073..1094)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1334..1353)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1681..1702
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1757..1837
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(2232..2248)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2232..2248)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2245..2267)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(2420..3008)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2500..2519)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(3125..3149)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(3249..3993,1..47,48..116))
                     /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     3872..3891
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
       61 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
      121 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
      181 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
      241 ccagatacct gaaacaaaac ccatcgtacg gccaaggaag tctccaataa ctgtgatcca
      301 ccacaagcgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tcatgcataa
      361 tccgcacgca tctggaataa ggaagtgcca ttccgcctga cctggagctt actggtgtca
      421 cttcttcacc tacttaataa ttcactgtta tcttcaagag gggatttgta ccattaaatg
      481 tagttacagt tttaagttgt taaatttgtt ataaatgact gctgaagttt tgacctttgc
      541 aatcatccct gtctgcagtg acacaggaat aattttaaac tgtcgagtga gataagatta
      601 tacatactgg gccacttacc tagggaactt ttagtagccc tgctttgatt atacttcctt
      661 tctcttgcag ctgtggacat cggaaaacca gggagacgaa ggcgatgccg gagagggaga
      721 gaacagtggt ggtacttccg ggggagggcc gagctctggc gcacccccac caagtggagg
      781 gtcacctgcc gggtccccaa catctactga agaaggcacc agcgaatccg caacgcccga
      841 gtcaggccct ggtacctcca cagaaccatc tgaaggtagt gcgcctggtt ccccagctgg
      901 aagccctact tccaccgaag aaggcacgtc aaccgaacca agtgaaggat ctgcccctgg
      961 gaccagcact gaaccatctg agggtggcgg attggaagtt ttgtttcaag gtccaggatc
     1021 taacggcagc gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     1081 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     1141 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     1201 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
     1261 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     1321 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     1381 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     1441 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     1501 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     1561 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     1621 caaccactac ctgagcaccc agtccaagct gagcaaagac cccaacgaga agcgcgatca
     1681 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     1741 caaggctgga gctggctacc catacgatgt tccagattac gcttatccgt atgacgtccc
     1801 ggactatgca tacccctacg atgtacccga ttacgccggt gcagggagtg gaagttaatg
     1861 tttctcgtgc tttgtgatct gttcagtgtc actctgtacc ctcaacatat atcccttgtg
     1921 cgataaaaaa aaaaaaaaaa aaaaaaagag aatcgtacgt cgactttcga tttttcacag
     1981 cctcagccta ggaaaaatgg ttcatgggat aaacagctgg tatttgtatc taaaactcag
     2041 attggtcaca taaatgccac ggcattccga agttttgatt ttgattaaca ttgacaggat
     2101 tactgtgtgt ttaatttttt aaaaactgaa cactgtgatt atggggtttt gtaatcgtca
     2161 gaagaccgtc agtgaaggct aggtggaggc tcagtgatga taagtctgcg atggtggatg
     2221 catgtgtcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcag agggcacaat
     2281 cctattccgc gctatccgac aatctccaag acattaggtg gagttcagtt cggcgtatgg
     2341 catatgtcgc tggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     2401 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2461 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2521 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2581 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     2641 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     2701 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     2761 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     2821 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     2881 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     2941 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3001 gatctcaaga agatcctttg atcttttcta cggggtctga cgctctattc aacaaagccg
     3061 ccgtcccgtc aagtcagcgt aaatgggtag ggggcttcaa atcgtcctcg tgataccaat
     3121 tcggagcctg cttttttgta caaacttgtt gataatggca attcaaggat cttcacctag
     3181 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     3241 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     3301 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     3361 tctggcccca gtgctgcaat gataccgcga gagccacgct caccggctcc agatttatca
     3421 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     3481 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     3541 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     3601 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     3661 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     3721 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     3781 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     3841 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     3901 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     3961 ttgagatcca gttcgatgta acccactcgt gca
//
