LOCUS       sequence-453970-        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: pML384_NCLN-C-term_XTEN80-mEGFP-3xHA_pTMPLT
FEATURES             Location/Qualifiers
     source          1..4893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..30
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(249..353)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     452..474
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     466..483
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     467..483
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1574..1597
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(1613..1615,1616..2326)
                     /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(1655..1676)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1916..1935)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2263..2284
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2339..2419
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(3264..3280)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3264..3280)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3277..3299)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(3452..4040)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3532..3551)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(4157..4181)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(4281..4893,1..179,180..248))
                     /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 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
       61 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
      121 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
      181 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
      241 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
      301 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
      361 ccccgaaaag tgccagatac ctgaaacaaa acccatcgta cggccaagga agtctccaat
      421 aactgtgatc caccacaagc gccagggttt tcccagtcac gacgttgtaa aacgacggcc
      481 agtcatgcat aatccgcacg catctggaat aaggaagtgc cattccgcct gacctggagc
      541 ttactggtgt cacttcctcg gactcctgag tagctgggat tacaggcacc cgccaccatg
      601 cccggctaat tttttttaat ttttagtaga gatggggttt caccgtgttg gccaggttgg
      661 tcttgaactc ttgacttcaa gtgatccgcc tgcctcaacc tcccaaagtg ctgggattgc
      721 aggtgtgagc catcgtgccc agtctccatc tctacaaacc cttttttaag aattagctgg
      781 gcgcagtggt gccccctgag aaagtgctct ctccccaggg acccagagtt tgtcttctac
      841 gaccagctga agcaagtgat gaatgcgtac aggtgagtgg tggccagcgg gacctggagc
      901 ccttcacccc ctacgggtta cagccgaggg gactgcggcc cacgggggtc taggggttca
      961 tgttactgcc gcgcaccatc ctcgacctca gggaccctgc tttctccaca gagtcaagcc
     1021 ggccgtcttt gacctgctcc tggctgttgg cattgctgcc tacctcggca tggcctacgt
     1081 ggctgtccag gtgagcagtg cccaggctca ggtggggcag gggccgcccg ccgggaggag
     1141 ctgggctggg cgtctccaag tgcatcctgg cccctggctc agcctagagt cacatgacct
     1201 ggggctctgg ccccgcccac atcctcactc cctcctgctg tgtccccagc acttcagcct
     1261 cctctacaag accgtccaga ggctgctcgt gaaggcaaag acacagagtg gtggtacttc
     1321 cgggggaggg ccgagctctg gcgcaccccc accaagtgga gggtcacctg ccgggtcccc
     1381 aacatctact gaagaaggca ccagcgaatc cgcaacgccc gagtcaggcc ctggtacctc
     1441 cacagaacca tctgaaggta gtgcgcctgg ttccccagct ggaagcccta cttccaccga
     1501 agaaggcacg tcaaccgaac caagtgaagg atctgcccct gggaccagca ctgaaccatc
     1561 tgagggtggc ggattggaag ttttgtttca aggtccagga tctaacggca gcgtgagcaa
     1621 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     1681 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     1741 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     1801 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     1861 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     1921 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     1981 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     2041 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     2101 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     2161 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     2221 ccagtccaag ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     2281 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaaggctg gagctggcta
     2341 cccatacgat gttccagatt acgcttatcc gtatgacgtc ccggactatg cataccccta
     2401 cgatgtaccc gattacgccg gtgcagggag tggaagttga cacagccacc cccacagccg
     2461 gagcccccgc cgctccacag tccctggggc cgagcacgag tgagtggaca ctgccccgcc
     2521 gcgggcggcc ctgcagggac aggggccctc tccctccccg gcggtggttg gaacactgaa
     2581 ttacagagct tttttctgtt gctctccgag actggggggg gattgtttct tcttttcctt
     2641 gtctttgaac ttccttggag gagagcttgg gagacgtccc ggggccaggc tacggacttg
     2701 cggacgagcc ccccagtcct gggagccggc cgccctcggt ctggtgtaag cacacatgca
     2761 cgattaaaga ggagacgccg ggaccccctg cccgatcgcg cgcggcctcc gcccaccgcc
     2821 tcctgccgca aggggcctgg actgcaggcc tgacctgctc cctgctccgt gtctgtccta
     2881 ggacgtcccc tcccgctccc cgatggtggc gtggacatgg ttatttatct ctgctccttc
     2941 ttgcctggag gagggcagtg ccagccctgg ggttctggga ttccagccct cctggagcct
     3001 tttgttcccc atgtggactc agtgacccgt ccccctgaca gtgggctcgg ggagctgcat
     3061 cacccagcct tccccttctc cgactgcagg gtctgatgtc atcattgaca gcctttgctt
     3121 cgtgggggcc tggcagggcc cctgcctccc cgacccccga cccactgcaa atccccgttc
     3181 ccctgcacgt cagaagaccg tcagtgaagg ctaggtggag gctcagtgat gataagtctg
     3241 cgatggtgga tgcatgtgtc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     3301 agagggcaca atcctattcc gcgctatccg acaatctcca agacattagg tggagttcag
     3361 ttcggcgtat ggcatatgtc gctggaaaga acatgtgagc aaaaggccag caaaaggcca
     3421 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     3481 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     3541 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     3601 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     3661 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     3721 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     3781 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     3841 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     3901 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     3961 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     4021 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctctat
     4081 tcaacaaagc cgccgtcccg tcaagtcagc gtaaatgggt agggggcttc aaatcgtcct
     4141 cgtgatacca attcggagcc tgcttttttg tacaaacttg ttgataatgg caattcaagg
     4201 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     4261 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     4321 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     4381 gagggcttac catctggccc cagtgctgca atgataccgc gagagccacg ctcaccggct
     4441 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     4501 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     4561 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     4621 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     4681 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     4741 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     4801 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     4861 tgtatgcggc gaccgagttg ctcttgcccg gcg
//
