LOCUS       sequence-452487-        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: pML365_HSP90AA1-N-term_XTEN80-mEGFP-3xHA_pTMPLT
FEATURES             Location/Qualifiers
     source          1..3993
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     22..41
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(260..364)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     463..485
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     477..494
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     478..494
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(724..735)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             886..966
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             join(979..981,982..1692)
                     /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(1021..1042)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1282..1301)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1629..1650
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1702..1725
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2375..2391)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2375..2391)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2388..2410)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(2563..3151)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2643..2662)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(3268..3292)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(3392..3993,1..190,191..259))
                     /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 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
       61 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
      121 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
      181 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
      241 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
      301 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
      361 cgcgcacatt tccccgaaaa gtgccagata cctgaaacaa aacccatcgt acggccaagg
      421 aagtctccaa taactgtgat ccaccacaag cgccagggtt ttcccagtca cgacgttgta
      481 aaacgacggc cagtcatgca taatccgcac gcatctggaa taaggaagtg ccattccgcc
      541 tgacctggag cttactggtg tcacttcccg ccgtggcgga gccgcagccc gcgcgggagg
      601 gcacccgggg tgttcgttgg ggaccgcggc gggggactgg gcccgggcct gcggcggcct
      661 cccggaagcg cgcacacgct cgtggtagtt gccgcgctcc gaaatgaggt catcctttgt
      721 cagccgccct tctattttcg gtttacttaa acttcgcaaa tctccaattc ggctttaaaa
      781 atatttggag aacgcgatgg aatttcgtgt tgcctctgta gacgtcctgc aaggttttaa
      841 accggcgcgg tgtcgttcca gatgcctagt ggtggtactt ccgggtaccc atacgatgtt
      901 ccagattacg cttatccgta tgacgtcccg gactatgcat acccctacga tgtacccgat
      961 tacgccgctg gagctggcgt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     1021 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     1081 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     1141 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     1201 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     1261 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     1321 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     1381 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     1441 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     1501 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     1561 ctgcccgaca accactacct gagcacccag tccaagctga gcaaagaccc caacgagaag
     1621 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     1681 gagctgtaca agggtggcgg attggaagtt ttgtttcaag gtccaggatc taacggcagc
     1741 ggagggccga gctctggcgc acccccacca agtggagggt cacctgccgg gtccccaaca
     1801 tctactgaag aaggcaccag cgaatccgca acgcccgagt caggccctgg tacctccaca
     1861 gaaccatctg aaggtagtgc gcctggttcc ccagctggaa gccctacttc caccgaagaa
     1921 ggcacgtcaa ccgaaccaag tgaaggatct gcccctggga ccagcactga accatctgag
     1981 ggtgcaggga gtggaagtga ggaaacccag acccaagacc aaccgatgga ggaggaggag
     2041 gttgagacgt tcgcctttca ggcagaaatt gcccagttga tgtcattgat catcaatact
     2101 ttctactcga acaaagagat ctttctgaga gagctcattt caaattcatc agatgtaagt
     2161 cacttattaa cccagaatcg gattttggtt tcagtgtgaa cttcttgggg gtgctgtatg
     2221 cttaaattaa tattttttgt taacaggcat tggacaaaat ccggtatgaa agcttgacag
     2281 atcccagtaa attagactcg tcagaagacc gtcagtgaag gctaggtgga ggctcagtga
     2341 tgataagtct gcgatggtgg atgcatgtgt catggtcata gctgtttcct gtgtgaaatt
     2401 gttatccgct cagagggcac aatcctattc cgcgctatcc gacaatctcc aagacattag
     2461 gtggagttca gttcggcgta tggcatatgt cgctggaaag aacatgtgag caaaaggcca
     2521 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     2581 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     2641 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     2701 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     2761 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     2821 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     2881 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     2941 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     3001 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     3061 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     3121 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     3181 tgacgctcta ttcaacaaag ccgccgtccc gtcaagtcag cgtaaatggg tagggggctt
     3241 caaatcgtcc tcgtgatacc aattcggagc ctgctttttt gtacaaactt gttgataatg
     3301 gcaattcaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     3361 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     3421 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     3481 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagagccac
     3541 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     3601 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     3661 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     3721 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     3781 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     3841 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     3901 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     3961 tctgagaata gtgtatgcgg cgaccgagtt gct
//
