LOCUS       sequence-452510-        4893 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 4893)
  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 4893)
  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: pML366_HSPA1B-N-term_XTEN80-mEGFP-3xHA_pTMPLT
FEATURES             Location/Qualifiers
     source          1..4893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..30
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(249..353)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     452..474
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     466..483
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     467..483
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1325..1405
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             join(1418..1420,1421..2131)
                     /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(1460..1481)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2068..2089
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2141..2164
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(3264..3280)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3264..3280)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3277..3299)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(3452..4040)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3532..3551)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(4157..4181)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(4281..4893,1..179,180..248))
                     /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 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
       61 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
      121 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
      181 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
      241 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
      301 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
      361 ccccgaaaag tgccagatac ctgaaacaaa acccatcgta cggccaagga agtctccaat
      421 aactgtgatc caccacaagc gccagggttt tcccagtcac gacgttgtaa aacgacggcc
      481 agtcatgcat aatccgcacg catctggaat aaggaagtgc cattccgcct gacctggagc
      541 ttactggtgt cacttcaaaa aacatccggg agagccggtc cgtttctcag gcagactagg
      601 ccattaggtg cctcggagaa aggacccaag gctgctccgt ccttcacaga cacagtccaa
      661 tcagagtttc ccaggcacat cgatgcaccg cctccttcga gaaacaaggt aactttcggg
      721 ttctggttgt ctccaaagtc atccgaccaa tctcgcaccg cccagagcgg gcccttcctg
      781 tcaattacct actgaagggc aggcggccag catcgccatg gagtccaaca cccttcccac
      841 caccactccc cctttctctc agggcccctg tcccctccag tgaatcccag aagactctgg
      901 agagttctga gcagagggcg gcaccctgcc ctctgattgg tccaaggaag gctggggggc
      961 aggacgggag gcgaaacccc tggaatattc ccgacctggc agcctcatcg agcttggtga
     1021 ttggctcaga aggggaaagg cgggactcca cgacgactta taaaagccga ggggcgcgcg
     1081 gtccggaaaa cggccagcct gaggagctgc tgcgagggtc cgcttcgtct ttcgagagtg
     1141 actcccgcgg tcccaaggct ttccagagcg aacctgtgcg gctgcaggca ccggcgtgtt
     1201 gagtttccgg cgttccgaag gactgagctc ttgtcgcgga tcccgtccgc cgtttccagc
     1261 ccccagtctc agagcggagc ccacagagca gggcaccggc atgtctagtg gtggtacttc
     1321 cgggtaccca tacgatgttc cagattacgc ttatccgtat gacgtcccgg actatgcata
     1381 cccctacgat gtacccgatt acgccgctgg agctggcgtg agcaagggcg aggagctgtt
     1441 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     1501 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     1561 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     1621 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     1681 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     1741 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     1801 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     1861 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     1921 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     1981 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccaagctgag
     2041 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     2101 gatcactctc ggcatggacg agctgtacaa gggtggcgga ttggaagttt tgtttcaagg
     2161 tccaggatct aacggcagcg gagggccgag ctctggcgca cccccaccaa gtggagggtc
     2221 acctgccggg tccccaacat ctactgaaga aggcaccagc gaatccgcaa cgcccgagtc
     2281 aggccctggt acctccacag aaccatctga aggtagtgcg cctggttccc cagctggaag
     2341 ccctacttcc accgaagaag gcacgtcaac cgaaccaagt gaaggatctg cccctgggac
     2401 cagcactgaa ccatctgagg gtgcagggag tggaagtgcc aaagccgcgg cgatcggcat
     2461 cgacctgggc accacctact cctgcgtggg ggtgttccaa cacggcaagg tggagatcat
     2521 cgccaacgac cagggcaacc gcaccacccc cagctacgtg gccttcacgg acaccgagcg
     2581 gctcatcggg gatgcggcca agaaccaggt ggcgctgaac ccgcagaaca ccgtgtttga
     2641 cgcgaagcgg ctgattggcc gcaagttcgg cgacccggtg gtgcagtcgg acatgaagca
     2701 ctggcctttc caggtgatca acgacggaga caagcccaag gtgcaggtga gctacaaggg
     2761 ggagacaaag gcattctacc ccgaggagat ctcgtccatg gtgctgacca agatgaagga
     2821 gatcgccgag gcgtacctgg gctacccggt gaccaacgcg gtgatcaccg tgccggccta
     2881 cttcaacgac tcgcagcgcc aggccaccaa ggatgcgggt gtgatcgcgg ggctcaacgt
     2941 gctgcggatc atcaacgagc ccacggccgc cgccatcgcc tacggcctgg acagaacggg
     3001 caagggggag cgcaacgtgc tcatctttga cctgggcggg ggcaccttcg acgtgtccat
     3061 cctgacgatc gacgacggca tcttcgaggt gaaggccacg gccggggaca cccacctggg
     3121 tggggaggac tttgacaaca ggctggtgaa ccacttcgtg gaggagttca agagaaaaca
     3181 caagaagcgt cagaagaccg tcagtgaagg ctaggtggag gctcagtgat gataagtctg
     3241 cgatggtgga tgcatgtgtc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     3301 agagggcaca atcctattcc gcgctatccg acaatctcca agacattagg tggagttcag
     3361 ttcggcgtat ggcatatgtc gctggaaaga acatgtgagc aaaaggccag caaaaggcca
     3421 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     3481 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     3541 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     3601 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     3661 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     3721 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     3781 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     3841 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     3901 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     3961 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     4021 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctctat
     4081 tcaacaaagc cgccgtcccg tcaagtcagc gtaaatgggt agggggcttc aaatcgtcct
     4141 cgtgatacca attcggagcc tgcttttttg tacaaacttg ttgataatgg caattcaagg
     4201 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     4261 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     4321 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     4381 gagggcttac catctggccc cagtgctgca atgataccgc gagagccacg ctcaccggct
     4441 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     4501 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     4561 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     4621 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     4681 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     4741 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     4801 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     4861 tgtatgcggc gaccgagttg ctcttgcccg gcg
//
