LOCUS       sequence-455737-        4892 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 4892)
  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 4892)
  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: pML346_VPS35-C-term_XTEN80-mEGFP-3xHA_pTMPLT
FEATURES             Location/Qualifiers
     source          1..4892
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     22..41
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(260..364)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     463..485
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     477..494
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     478..494
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1585..1608
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(1624..1626,1627..2337)
                     /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(1666..1687)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1927..1946)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2274..2295
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2350..2430
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(3274..3290)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3274..3290)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3287..3309)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(3462..4050)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3542..3561)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(4167..4191)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(4291..4892,1..190,191..259))
                     /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 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
       61 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
      121 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
      181 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
      241 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
      301 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
      361 cgcgcacatt tccccgaaaa gtgccagata cctgaaacaa aacccatcgt acggccaagg
      421 aagtctccaa taactgtgat ccaccacaag cgccagggtt ttcccagtca cgacgttgta
      481 aaacgacggc cagtcatgca taatccgcac gcatctggaa taaggaagtg ccattccgcc
      541 tgacctggag cttactggtg tcacttccct aaacgcagta tccttttagc aatacagttt
      601 tggtcaagat ttgtaagtta aataaaattt tgcttgtttt tctttttttg acagagtctg
      661 gctctgtcac ccaggctgga gtgcagtggc aatctcagct cattgcaacc tctgcctccc
      721 aggttcaagc aattctcatg cctcagcctc ctgaatagct ggtattatag gcgcccggca
      781 ccacgcccag ctaatttttg tattattact agagatgggg ttccaccatg ttggccaggc
      841 tggtctgaaa actcctgact tcaagtgatc tgcccacctc agcctcccaa agtgctggga
      901 gtacagggat gagccactga gccgagccaa ttttgcttgt tttaaagggt tgtttttttt
      961 tttttttttt tgatagtcag taattgttca aactaggaat tgtatcccca tctttctttt
     1021 ttcataatta ctcaggtaat tgatgagtgt aacagaagct cctcaaaaca gttttattaa
     1081 attgcctttc attttttgtg gtacgtgctt gatcatgaat ttgtacatat tcttttgtag
     1141 gtaacaattc aggttttaaa ccagcttatc caaaagattc gagaagacct cccgaatctt
     1201 gaatccagtg aagaaacaga gcagattaac aaacattttc ataacacact ggagcatttg
     1261 cgcttgcggc gggaatcacc agaatccgag gggccaattt atgaaggtct gatccttagt
     1321 ggtggtactt ccgggggagg gccgagctct ggcgcacccc caccaagtgg agggtcacct
     1381 gccgggtccc caacatctac tgaagaaggc accagcgaat ccgcaacgcc cgagtcaggc
     1441 cctggtacct ccacagaacc atctgaaggt agtgcgcctg gttccccagc tggaagccct
     1501 acttccaccg aagaaggcac gtcaaccgaa ccaagtgaag gatctgcccc tgggaccagc
     1561 actgaaccat ctgagggtgg cggattggaa gttttgtttc aaggtccagg atctaacggc
     1621 agcgtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     1681 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     1741 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     1801 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     1861 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     1921 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     1981 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     2041 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     2101 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     2161 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     2221 tacctgagca cccagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     2281 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaaggct
     2341 ggagctggct acccatacga tgttccagat tacgcttatc cgtatgacgt cccggactat
     2401 gcatacccct acgatgtacc cgattacgcc ggtgcaggga gtggaagtta aaaaggaaat
     2461 agctcaccat actcctttcc atgtacatcc agtgagggtt ttattacgct aggtttccct
     2521 tccatagatt gtgcctttca gaaatgctga ggtaggtttc ccatttctta cctgtgatgt
     2581 gttttaccca gcacctccgg acactcacct tcaggacctt aataaaatta ttcacttggt
     2641 aagtgttcaa gtctttctga tcaccccaag tagcatgact gatctgcaat ttaaaattcc
     2701 tgtgatctgt aaaaaaaaaa aaaaaaaaaa cacaaaaccc acaagcactt atcttggcta
     2761 ctaatgaagc tctccttttt tttgtttgtt tgtttgcttc attgttgatt gtgtattttc
     2821 ttcattcctg gggagtacta acccaaaagc gtctgtctct tgttttctag tccagtttga
     2881 gattaattta gaagaaagga atactgtatg tgaaattcat cttgggcttt cccctaaatt
     2941 gcaagataag gccatgtgta agattttccc taaaactaga atatattaat gcatgtttga
     3001 gaattttaaa gcaccatggt caaaaccaga agctatattt tgcatatttg gactcagcca
     3061 tccattaaga acccatgttg tcctctggac atatttatca atataattgg gttttaaata
     3121 gtataaaaga aaacttgtga tctatataat ttatgtatca ccttcattgt aaatttagca
     3181 ggaaatgcat cacaattcgt cagaagaccg tcagtgaagg ctaggtggag gctcagtgat
     3241 gataagtctg cgatggtgga tgcatgtgtc atggtcatag ctgtttcctg tgtgaaattg
     3301 ttatccgctc agagggcaca atcctattcc gcgctatccg acaatctcca agacattagg
     3361 tggagttcag ttcggcgtat ggcatatgtc gctggaaaga acatgtgagc aaaaggccag
     3421 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     3481 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     3541 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     3601 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     3661 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     3721 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     3781 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     3841 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     3901 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     3961 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4021 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4081 gacgctctat tcaacaaagc cgccgtcccg tcaagtcagc gtaaatgggt agggggcttc
     4141 aaatcgtcct cgtgatacca attcggagcc tgcttttttg tacaaacttg ttgataatgg
     4201 caattcaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     4261 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     4321 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     4381 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagagccacg
     4441 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     4501 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     4561 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     4621 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     4681 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     4741 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     4801 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     4861 ctgagaatag tgtatgcggc gaccgagttg ct
//
