LOCUS       sequence-456010-        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: pML323_GOLGA2-N-term_XTEN80-mEGFP-3xHA_pTMPLT
FEATURES             Location/Qualifiers
     source          1..3993
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(113..217)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     316..338
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     330..347
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     331..347
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             739..819
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             join(832..834,835..1545)
                     /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(874..895)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1135..1154)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1482..1503
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1555..1578
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2228..2244)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2228..2244)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2241..2263)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(2416..3004)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2496..2515)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(3121..3145)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(3245..3993,1..43,44..112))
                     /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     3868..3887
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
       61 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
      121 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
      181 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccag
      241 atacctgaaa caaaacccat cgtacggcca aggaagtctc caataactgt gatccaccac
      301 aagcgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtcat gcataatccg
      361 cacgcatctg gaataaggaa gtgccattcc gcctgacctg gagcttactg gtgtcacttc
      421 cgcacagaaa gctgcgcgcg cacccaagga aagccggccc cgcccctctc ccgaggtggg
      481 cggggacagc ggttgcgtgc gcagcttttg ttgtgacgcc acaggtccct ctggccacgc
      541 cgctgcatag ccacgccccc ttccgcttca tatttctcat tgaccaatgg gctttgatct
      601 ttgaggccac gcccccattc tgcgctccag tatcgccctg gttacgcctc ctctggctcc
      661 gtcgcacagc tgcgcggtgg ctgactgact tccgcagtgg ttgccgggat cgcgcacatg
      721 agtggtggta cttccgggta cccatacgat gttccagatt acgcttatcc gtatgacgtc
      781 ccggactatg cataccccta cgatgtaccc gattacgccg ctggagctgg cgtgagcaag
      841 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
      901 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
      961 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1021 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     1081 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     1141 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1201 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1261 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     1321 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     1381 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     1441 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     1501 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagggtgg cggattggaa
     1561 gttttgtttc aaggtccagg atctaacggc agcggagggc cgagctctgg cgcaccccca
     1621 ccaagtggag ggtcacctgc cgggtcccca acatctactg aagaaggcac cagcgaatcc
     1681 gcaacgcccg agtcaggccc tggtacctcc acagaaccat ctgaaggtag tgcgcctggt
     1741 tccccagctg gaagccctac ttccaccgaa gaaggcacgt caaccgaacc aagtgaagga
     1801 tctgcccctg ggaccagcac tgaaccatct gagggtgcag ggagtggaag ttggccccaa
     1861 ccccgcctcc ctccccgccc cgcgatgtcg gaagaaaccc gacagagcaa attggccgca
     1921 gcgaagaaaa aggtaaagtg caccgggtcg cggccccccg acccagcccc agccccactc
     1981 cgatggcagg accatggcca gagtctctgc cactcctgag gcacaccggg ctgggctccc
     2041 cacggcgcgt ccgggctccc tccaccaaag tcttgtcagc cagccccgcc ccctcagccg
     2101 cccagccccc gccctcgcca gtcgcccccg ggtgactttg ggctggtgct ccgtcagaag
     2161 accgtcagtg aaggctaggt ggaggctcag tgatgataag tctgcgatgg tggatgcatg
     2221 tgtcatggtc atagctgttt cctgtgtgaa attgttatcc gctcagaggg cacaatccta
     2281 ttccgcgcta tccgacaatc tccaagacat taggtggagt tcagttcggc gtatggcata
     2341 tgtcgctgga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     2401 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     2461 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     2521 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     2581 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     2641 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     2701 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     2761 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     2821 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     2881 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     2941 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     3001 tcaagaagat cctttgatct tttctacggg gtctgacgct ctattcaaca aagccgccgt
     3061 cccgtcaagt cagcgtaaat gggtaggggg cttcaaatcg tcctcgtgat accaattcgg
     3121 agcctgcttt tttgtacaaa cttgttgata atggcaattc aaggatcttc acctagatcc
     3181 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     3241 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     3301 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     3361 gccccagtgc tgcaatgata ccgcgagagc cacgctcacc ggctccagat ttatcagcaa
     3421 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     3481 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     3541 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     3601 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     3661 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     3721 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3781 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     3841 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     3901 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     3961 gatccagttc gatgtaaccc actcgtgcac cca
//
