LOCUS       sequence-452494-        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: pML374_CLTA-N-term_XTEN80-mEGFP-3xHA_pTMPLT
FEATURES             Location/Qualifiers
     source          1..4893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     437..456
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(675..779)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     878..900
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     892..909
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     893..909
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1751..1831
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             join(1844..1846,1847..2557)
                     /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(1886..1907)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2147..2166)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2494..2515
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2567..2590
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(3690..3706)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3690..3706)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3703..3725)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(3878..4466)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3958..3977)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(4583..4607)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(4707..4893,1..605,606..674))
                     /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 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
       61 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
      121 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
      181 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
      241 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
      301 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
      361 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
      421 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
      481 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
      541 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
      601 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
      661 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
      721 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
      781 acatttcccc gaaaagtgcc agatacctga aacaaaaccc atcgtacggc caaggaagtc
      841 tccaataact gtgatccacc acaagcgcca gggttttccc agtcacgacg ttgtaaaacg
      901 acggccagtc atgcataatc cgcacgcatc tggaataagg aagtgccatt ccgcctgacc
      961 tggagcttac tggtgtcact tcaaaaatac ttatgggcga gaaaaatcgg agtggactga
     1021 atcaattaca gtaaatctat gggctctaag ggagcaaata atccaggctg gctgaaagaa
     1081 ttaaatattt taaatttata aagccaggga ccctcaaatc cataaaagga ttcaatcttc
     1141 ttcttctttt tttttaggat tcaatcttct aataatcccg ggagggggcg caagagcagg
     1201 attcttgttt ctattttacg aatgaggaaa ctgaggttca gaaagcctaa gtggcatgcc
     1261 tcttcagtaa tcggccaatc gtcgctgccc taacttcacc tcacctccca gtgcagcact
     1321 agagtcccct caggggtggc cgtccctggt cagtccagct ccagcagacg gggtgggtag
     1381 gacgctccct atcaggcagc acttccgcct cccggggccc gcgcagctca cctccctcac
     1441 ctcccgccct accccagtca cgagttgttt tagggggacc gcccctccac ttgctgattg
     1501 ggtagctcct gaaccattgt tgtcctctga ttggttgttc ccttttcggc tctgcaacac
     1561 cgcctagacc gaccggatac acgggtaggg cttccgcttt acccgtctcc ctcctggcgc
     1621 ttgtcctcct ctcccagtcg gcaccacagc ggtggctgcc gggcgtggtg tcggtgggtc
     1681 ggttggtttt tgtctcaccg ttggtgtccg tgccgttcag ttgcccgcca tgagtggtgg
     1741 tacttccggg tacccatacg atgttccaga ttacgcttat ccgtatgacg tcccggacta
     1801 tgcatacccc tacgatgtac ccgattacgc cgctggagct ggcgtgagca agggcgagga
     1861 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     1921 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     1981 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     2041 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     2101 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     2161 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     2221 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     2281 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     2341 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     2401 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccaa
     2461 gctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     2521 cgccgggatc actctcggca tggacgagct gtacaagggt ggcggattgg aagttttgtt
     2581 tcaaggtcca ggatctaacg gcagcggagg gccgagctct ggcgcacccc caccaagtgg
     2641 agggtcacct gccgggtccc caacatctac tgaagaaggc accagcgaat ccgcaacgcc
     2701 cgagtcaggc cctggtacct ccacagaacc atctgaaggt agtgcgcctg gttccccagc
     2761 tggaagccct acttccaccg aagaaggcac gtcaaccgaa ccaagtgaag gatctgcccc
     2821 tgggaccagc actgaaccat ctgagggtgc agggagtgga agtgctgagc tggatccgtt
     2881 cggcgcccct gccggcgccc ctggcggtcc cgcgctgggg aacggagtgg ccggcgccgg
     2941 cgaagaagac ccggctgcgg ccttcttggc gcagcaagag agcgagattg cgggcatcga
     3001 gaacgacgag gccttcgcca tcctggacgg cggcgccccc gggccccagc cgcacggcga
     3061 gccgccgggg ggtccgggtg agagtgcggg cgcgtttggg gcgagaggac ttgtctggaa
     3121 actcggtcca cagtgggtcc gagagcttct gtgtgactcg tgctccttgc tgaattagga
     3181 ggttagggag cagtgcaaac aggaaacgtg accctggccc ggtctttcag aaacctaggc
     3241 tcgagaagcc tgttcggttc tcagcatgtt tgagtgcttc tgggcgcggg cggagcgaga
     3301 aagcaagtgt agggtggcag gctccggagc cggaagaagc ccgttcaatt cagcaacttt
     3361 tcattaagca tttgctgtgc ctttagtccg gtcactgaag caaccgcatt ggcgcagttt
     3421 ttccagactt ataagcttat aagtctgagc cgagcacaga actcgttatt agaaaaggag
     3481 ggcggaaaaa aataagaatg gaaatatcgt ttttgagaga tacaaacaaa agtagcaatg
     3541 cagttcagca tttaagcact taaggtgtac agagtgttgg attacgagga ggaaggaggg
     3601 agtagggaac tcgcgtcaga agaccgtcag tgaaggctag gtggaggctc agtgatgata
     3661 agtctgcgat ggtggatgca tgtgtcatgg tcatagctgt ttcctgtgtg aaattgttat
     3721 ccgctcagag ggcacaatcc tattccgcgc tatccgacaa tctccaagac attaggtgga
     3781 gttcagttcg gcgtatggca tatgtcgctg gaaagaacat gtgagcaaaa ggccagcaaa
     3841 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     3901 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     3961 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4021 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4081 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4141 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4201 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     4261 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     4321 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     4381 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     4441 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     4501 ctctattcaa caaagccgcc gtcccgtcaa gtcagcgtaa atgggtaggg ggcttcaaat
     4561 cgtcctcgtg ataccaattc ggagcctgct tttttgtaca aacttgttga taatggcaat
     4621 tcaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4681 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4741 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     4801 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga gccacgctca
     4861 ccggctccag atttatcagc aataaaccag cca
//
