LOCUS       sequence-452481-        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: pML319_SEC31A-N-term_XTEN80-mEGFP-3xHA_pTMPLT
FEATURES             Location/Qualifiers
     source          1..3993
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(116..220)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     319..341
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     333..350
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     334..350
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             742..822
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             join(835..837,838..1548)
                     /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(877..898)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1138..1157)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1485..1506
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1558..1581
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2231..2247)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2231..2247)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2244..2266)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(2419..3007)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2499..2518)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(3124..3148)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(3248..3993,1..46,47..115))
                     /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     3871..3890
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
       61 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
      121 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
      181 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
      241 cagatacctg aaacaaaacc catcgtacgg ccaaggaagt ctccaataac tgtgatccac
      301 cacaagcgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt catgcataat
      361 ccgcacgcat ctggaataag gaagtgccat tccgcctgac ctggagctta ctggtgtcac
      421 ttcgatgggg tgtcaccatg ttagccagga tggtgtcaat ctcctgacct cgtgatccgc
      481 ccgccttggc ctcccaaagt gctgggatta caggcatgag ccacccggcc tcaagtttgt
      541 cttctttcta atgaatatat ccaacaaatg acacctaaac cgatttgatt tatttgcagt
      601 tgggcattac taatgttgta gtggtttgtt cagtatgttt ctatttaaaa tatctgtgtt
      661 ctgtgttaga cattcaagtc attcaaggct gtttctgtat aaaatattcc ctttagcagg
      721 atgagtggtg gtacttccgg gtacccatac gatgttccag attacgctta tccgtatgac
      781 gtcccggact atgcataccc ctacgatgta cccgattacg ccgctggagc tggcgtgagc
      841 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
      901 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
      961 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     1021 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     1081 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     1141 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     1201 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     1261 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     1321 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     1381 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     1441 acccagtcca agctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     1501 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaaggg tggcggattg
     1561 gaagttttgt ttcaaggtcc aggatctaac ggcagcggag ggccgagctc tggcgcaccc
     1621 ccaccaagtg gagggtcacc tgccgggtcc ccaacatcta ctgaagaagg caccagcgaa
     1681 tccgcaacgc ccgagtcagg ccctggtacc tccacagaac catctgaagg tagtgcgcct
     1741 ggttccccag ctggaagccc tacttccacc gaagaaggca cgtcaaccga accaagtgaa
     1801 ggatctgccc ctgggaccag cactgaacca tctgagggtg cagggagtgg aagtaagtta
     1861 aaggaagtag atcgtacagc catgcaggca tggagccctg cccagaatca ccccatttac
     1921 ctagcaacag gtaagttcaa aggagagaag atagtatgag ttacctaatt tcttctcttc
     1981 tgcacctagc ttattcagtt taagctttat cttctctaga aagcctctct ctccctggca
     2041 agttactgac attctatcag ccaagtttag tgttcttcct ctgtgttccc atagcatttt
     2101 gatcatgatg tgttacatct aaaatatttt ccttcacagg aattgtattt tatccgtcag
     2161 aagaccgtca gtgaaggcta ggtggaggct cagtgatgat aagtctgcga tggtggatgc
     2221 atgtgtcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaga gggcacaatc
     2281 ctattccgcg ctatccgaca atctccaaga cattaggtgg agttcagttc ggcgtatggc
     2341 atatgtcgct ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     2401 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     2461 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     2521 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     2581 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     2641 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     2701 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     2761 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     2821 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     2881 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     2941 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     3001 atctcaagaa gatcctttga tcttttctac ggggtctgac gctctattca acaaagccgc
     3061 cgtcccgtca agtcagcgta aatgggtagg gggcttcaaa tcgtcctcgt gataccaatt
     3121 cggagcctgc ttttttgtac aaacttgttg ataatggcaa ttcaaggatc ttcacctaga
     3181 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     3241 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     3301 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     3361 ctggccccag tgctgcaatg ataccgcgag agccacgctc accggctcca gatttatcag
     3421 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     3481 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     3541 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     3601 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     3661 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     3721 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     3781 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     3841 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     3901 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     3961 tgagatccag ttcgatgtaa cccactcgtg cac
//
