LOCUS       sequence-468625-        4393 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 4393)
  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 4393)
  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: pML625_RTN4-N-term_XTEN80-mEGFP-3xHA_pTMPLT
FEATURES             Location/Qualifiers
     source          1..4393
                     /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             1078..1158
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             join(1171..1173,1174..1884)
                     /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(1213..1234)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1474..1493)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1821..1842
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1894..1917
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2767..2783)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2767..2783)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2780..2802)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(2955..3543)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3035..3054)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(3660..3684)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(3784..4393,1..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
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     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 cccaccccaa ctcacggtta cccaaagtgg gggtggagct
      601 cccagctctc agccccgccc attgaacgca atcccagccg gtaagagaga ggaagtgccg
      661 tagtccccac ccactttcat ctccttccct ttctgttggc ccggctggtc gcctatcttc
      721 tcgatcctgt agccgattgg ctctctccct cccgcgccag tccctgccct cccctgggga
      781 gggtgagtca cgccaaactg ggcggagagt ccgctggcct cactcctagc tcatctgggc
      841 ggcggcggca agtggggaca gggcgggtgg cgcatcaccg gcgcggaggc aggaggagca
      901 gtctcattgt tccgggagcc gtcaccacag taggtccctc ggctcagtcg gcccagcccc
      961 tctcagtcct ccccaacccc cacaaccgcc cgcggctctg agacgcggcc ccggcggcgg
     1021 cggcagcagc tgcagcatca tctccaccct ccagccatgt ctggtggtac ttccgggtac
     1081 ccatacgatg ttccagatta cgcttatccg tatgacgtcc cggactatgc atacccctac
     1141 gatgtacccg attacgccgc tggagctggc gtgagcaagg gcgaggagct gttcaccggg
     1201 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     1261 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     1321 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
     1381 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     1441 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     1501 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     1561 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
     1621 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     1681 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     1741 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccaagct gagcaaagac
     1801 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     1861 ctcggcatgg acgagctgta caagggtggc ggattggaag ttttgtttca aggtccagga
     1921 tctaacggca gcggagggcc gagctctggc gcacccccac caagtggagg gtcacctgcc
     1981 gggtccccaa catctactga agaaggcacc agcgaatccg caacgcccga gtcaggccct
     2041 ggtacctcca cagaaccatc tgaaggtagt gcgcctggtt ccccagctgg aagccctact
     2101 tccaccgaag aaggcacgtc aaccgaacca agtgaaggat ctgcccctgg gaccagcact
     2161 gaaccatctg agggtgcagg gagtggaagt gaagacctgg accagtctcc tctggtctcg
     2221 tcctcggaca gcccaccccg gccgcagccc gcgttcaagt accagttcgt gagggagccc
     2281 gaggacgagg aggaagaaga ggaggaggaa gaggaggacg aggacgaaga cctggaggag
     2341 ctggaggtgc tggagaggaa gcccgccgcc gggctgtccg cggccccagt gcccaccgcc
     2401 cctgccgccg gcgcgcccct gatggacttc ggaaatgact tcgtgccgcc ggcgccccgg
     2461 ggacccctgc cggccgctcc ccccgtcgcc ccggagcggc agccgtcttg ggacccgagc
     2521 ccggtgtcgt cgaccgtgcc cgcgccatcc ccgctgtctg ctgccgcagt ctcgccctcc
     2581 aagctccctg aggacgacga gcctccggcc cggcctcccc ctcctccccc ggccagcgtg
     2641 agcccccagg cagagcccgt gtggaccccg ccagccccgg ctcccgccgc cgtcagaaga
     2701 ccgtcagtga aggctaggtg gaggctcagt gatgataagt ctgcgatggt ggatgcatgt
     2761 gtcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcagagggc acaatcctat
     2821 tccgcgctat ccgacaatct ccaagacatt aggtggagtt cagttcggcg tatggcatat
     2881 gtcgctggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     2941 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     3001 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     3061 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     3121 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     3181 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     3241 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     3301 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     3361 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     3421 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     3481 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     3541 caagaagatc ctttgatctt ttctacgggg tctgacgctc tattcaacaa agccgccgtc
     3601 ccgtcaagtc agcgtaaatg ggtagggggc ttcaaatcgt cctcgtgata ccaattcgga
     3661 gcctgctttt ttgtacaaac ttgttgataa tggcaattca aggatcttca cctagatcct
     3721 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     3781 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     3841 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     3901 ccccagtgct gcaatgatac cgcgagagcc acgctcaccg gctccagatt tatcagcaat
     3961 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     4021 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     4081 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     4141 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     4201 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     4261 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     4321 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     4381 ttgctcttgc ccg
//
