LOCUS       sequence-455749-        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: pML348_ATG101-N-term_XTEN80-mEGFP-3xHA_pTMPLT
FEATURES             Location/Qualifiers
     source          1..4893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     14..33
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(252..356)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     455..477
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     469..486
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     470..486
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1328..1408
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             join(1421..1423,1424..2134)
                     /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(1463..1484)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1724..1743)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2071..2092
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2144..2167
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(3267..3283)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3267..3283)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3280..3302)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(3455..4043)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3535..3554)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(4160..4184)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(4284..4893,1..182,183..251))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGEILTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
       61 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      121 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      181 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
      241 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
      301 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
      361 tttccccgaa aagtgccaga tacctgaaac aaaacccatc gtacggccaa ggaagtctcc
      421 aataactgtg atccaccaca agcgccaggg ttttcccagt cacgacgttg taaaacgacg
      481 gccagtcatg cataatccgc acgcatctgg aataaggaag tgccattccg cctgacctgg
      541 agcttactgg tgtcacttcc tgggactaca ggcgcctgcc accaggccca gctaattttt
      601 tatatttttg gtagagatgg ggttttatca tgtttcccag gctggtcttg aacttttgag
      661 ctcaagtgat ttgcctcggc ctcccaagtt gctgggatta atgtgagcca ccacacccgg
      721 cctagaggta cttttttgat gcacagattt gatcttggct tagtgatgca ttgactcttc
      781 gctgctttta ggacagagtc caaatgcctt aacctgttca ttcaaggtcc caggaaatca
      841 gactccaatc tttctgtact tcttactccc accctgtact ctctccgtgg tgataatgtt
      901 tagttgcgtg ctttttcttc atcctgtcag cctgagggca ggggccgtgc ttgcctttga
      961 tgtctagtac ctgacaaatg ttgcacagtg agcatctgct gtaaactaca ttgaaggctg
     1021 taaaatggaa tcccagcctg tcatttggac tgataacttt gaagctcttg cactttctga
     1081 tcctgagact tgatgattcg ttgatagtgt gtgctgccaa aatgcagaga agggccagtg
     1141 gaggcagagg ctgagctgag cctagaagga agtgaacaga taacacgtgg actattcttg
     1201 tcagcattct gacctgggct ccttttgcag ggtggccctt gggtagggtg aagatcccct
     1261 gtctttatcc tagttccaca ccttggtgtg ggttactggg tgcaggatga gtggtggtac
     1321 ttccgggtac ccatacgatg ttccagatta cgcttatccg tatgacgtcc cggactatgc
     1381 atacccctac gatgtacccg attacgccgc tggagctggc gtgagcaagg gcgaggagct
     1441 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     1501 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     1561 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
     1621 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     1681 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     1741 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     1801 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     1861 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     1921 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     1981 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccaagct
     2041 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     2101 cgggatcact ctcggcatgg acgagctgta caagggtggc ggattggaag ttttgtttca
     2161 aggtccagga tctaacggca gcggagggcc gagctctggc gcacccccac caagtggagg
     2221 gtcacctgcc gggtccccaa catctactga agaaggcacc agcgaatccg caacgcccga
     2281 gtcaggccct ggtacctcca cagaaccatc tgaaggtagt gcgcctggtt ccccagctgg
     2341 aagccctact tccaccgaag aaggcacgtc aaccgaacca agtgaaggat ctgcccctgg
     2401 gaccagcact gaaccatctg agggtgcagg gagtggaagt aactgtcgct ctgaggtgct
     2461 ggaggtgtcg gtggaggggc ggcaggtgga ggaggccatg ctggctgtgc tgcacacggt
     2521 gcttctgcac cgcagcacag gcaagttcca ctacaagaag gagggcacct actccattgg
     2581 caccgtgggc acccaggatg ttgactgtga cttcatcgac ttcacttatg tgcgtgtctc
     2641 ttctgaggaa ctggatcgtg ccctgcgcaa ggttgttggg gagttcaagg taagggtgtc
     2701 ggggagttca aggtaagggt gtggcatagc agatggcagg ggggcctcga gtgctcctcc
     2761 actccagctt ggtgttgaac cagccttctc ttcctgagtg cgtgaatcgg gttctttcag
     2821 gttctgagaa acagatgtgt tgagagctcc tcttcagggg agggcgcgcg ggcgcgtgtg
     2881 tgtgtggtgg gggagggaga gactcataaa tgtgcctggg ggaaaaggga gagcaggaag
     2941 ctgagcagtg ttggcggcca ggtcccctgg ggccatcctt gcttctgtgg tcttgacctc
     3001 tctggttaca gctctctctg cttctgcttc cacttttcct ttcccacatg aaactccccc
     3061 agaggacggc ttgcttggtt caggtggctg ccatccaaga tgggagtgtc ccttttggtg
     3121 gtgtggcatt gtgtgcctgg gtaggagaaa gatgtagaag ggacacggcc ggctgctgct
     3181 gttggtcacc cgtcagaaga ccgtcagtga aggctaggtg gaggctcagt gatgataagt
     3241 ctgcgatggt ggatgcatgt gtcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     3301 ctcagagggc acaatcctat tccgcgctat ccgacaatct ccaagacatt aggtggagtt
     3361 cagttcggcg tatggcatat gtcgctggaa agaacatgtg agcaaaaggc cagcaaaagg
     3421 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3481 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3541 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3601 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3661 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3721 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     3781 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     3841 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     3901 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     3961 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4021 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4081 tattcaacaa agccgccgtc ccgtcaagtc agcgtaaatg ggtagggggc ttcaaatcgt
     4141 cctcgtgata ccaattcgga gcctgctttt ttgtacaaac ttgttgataa tggcaattca
     4201 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     4261 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     4321 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     4381 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagagcc acgctcaccg
     4441 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     4501 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtatt
     4561 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     4621 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     4681 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     4741 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     4801 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     4861 tagtgtatgc ggcgaccgag ttgctcttgc ccg
//
