LOCUS       sequence-456021-        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: pML357_CAV1-C-term_XTEN80-mEGFP-3xHA_pTMPLT
FEATURES             Location/Qualifiers
     source          1..4893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             747..770
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(786..788,789..1499)
                     /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(828..849)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1089..1108)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1436..1457
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1512..1592
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(2437..2453)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2437..2453)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2450..2472)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(2625..3213)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2705..2724)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(3330..3354)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(3454..4245,4246..4314))
                     /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     4077..4096
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4315..4419)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4518..4540
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4532..4549
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4533..4549
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gctgctggtt gcctttaagc atctcacaaa gaacgaactc ataaatgcta atacagtata
       61 tgtctatgga tactgaatag tgggtttttt ttctcttttc ttctattctg tgctcatgtt
      121 gtgtcacttc ttccttttag attgactttg aagatgtgat tgcagaacca gaagggacac
      181 acagttttga cggcatttgg aaggccagct tcaccacctt cactgtgacg aaatactggt
      241 tttaccgctt gctgtctgcc ctctttggca tcccgatggc actcatctgg ggcatttact
      301 tcgccattct ctctttcctg cacatctggg cagttgtacc atgcattaag agcttcctga
      361 ttgagattca gtgcatcagc cgtgtctatt ccatctacgt ccacaccgtc tgtgacccac
      421 tctttgaagc tgttgggaaa atattcagca atgtacgcat caacttgcag aaagaaataa
      481 gtggtggtac ttccggggga gggccgagct ctggcgcacc cccaccaagt ggagggtcac
      541 ctgccgggtc cccaacatct actgaagaag gcaccagcga atccgcaacg cccgagtcag
      601 gccctggtac ctccacagaa ccatctgaag gtagtgcgcc tggttcccca gctggaagcc
      661 ctacttccac cgaagaaggc acgtcaaccg aaccaagtga aggatctgcc cctgggacca
      721 gcactgaacc atctgagggt ggcggattgg aagttttgtt tcaaggtcca ggatctaacg
      781 gcagcgtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
      841 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
      901 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
      961 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     1021 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     1081 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     1141 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     1201 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     1261 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     1321 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     1381 actacctgag cacccagtcc aagctgagca aagaccccaa cgagaagcgc gatcacatgg
     1441 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg
     1501 ctggagctgg ctacccatac gatgttccag attacgctta tccgtatgac gttccggact
     1561 atgcataccc ctacgatgta cccgattacg ccggtgcagg gagtggaagt taaatgacat
     1621 ttcaaggata gaagtatacc tgattttttt tccttttaat tttcctggtg ccaatttcaa
     1681 gttccaagtt gctaatacag caacaattta tgaattgaat tatcttggtt gaaaataaaa
     1741 agatcacttt ctcagttttc ataagtatta tgtctcttct gagctatttc atctattttt
     1801 ggcagtctga atttttaaaa cccatttaaa tttttttcct taccttttta tttgcatgtg
     1861 gatcaaccat cgctttattg gctgagatat gaacatattg ttgaaaggta atttgagaga
     1921 aatatgaaga actgaggagg aaaaaaaaaa aaaagaaaag aaccaacaac ctcaactgcc
     1981 tactccaaaa tgttggtcat tttatgttaa gggaagaatt ccagggtatg gccatggagt
     2041 gtacaagtat gtgggcagat tttcagcaaa ctcttttccc actgtttaag gagttagtgg
     2101 attactgcca ttcacttcat aatccagtag gatccagtga tccttacaag ttagaaaaca
     2161 taatcttctg ccttctcatg atccaactaa tgccttactc ttcttgaaat tttaacctat
     2221 gatattttct gtgcctgaat atttgttatg tagataacaa gacctcagtg ccttcctgtt
     2281 tttcacattt tccttttcaa atagggtcta actcagcaac tcgctttagg tcagcagcct
     2341 ccctgaagac caaaattaga cgtcagaaga ccgtcagtga aggctaggtg gaggctcagt
     2401 gatgataagt ctgcgatggt ggatgcatgt gtcatggtca tagctgtttc ctgtgtgaaa
     2461 ttgttatccg ctcagagggc acaatcctat tccgcgctat ccgacaatct ccaagacatt
     2521 aggtggagtt cagttcggcg tatggcatat gtcgctggaa agaacatgtg agcaaaaggc
     2581 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     2641 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     2701 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     2761 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     2821 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     2881 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     2941 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     3001 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     3061 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3121 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3181 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     3241 tctgacgctc tattcaacaa agccgccgtc ccgtcaagtc agcgtaaatg ggtagggggc
     3301 ttcaaatcgt cctcgtgata ccaattcgga gcctgctttt ttgtacaaac ttgttgataa
     3361 tggcaattca aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     3421 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     3481 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     3541 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagagcc
     3601 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     3661 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     3721 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     3781 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     3841 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     3901 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     3961 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     4021 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     4081 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     4141 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     4201 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     4261 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     4321 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     4381 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     4441 agatacctga aacaaaaccc atcgtacggc caaggaagtc tccaataact gtgatccacc
     4501 acaagcgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtc atgcataatc
     4561 cgcacgcatc tggaataagg aagtgccatt ccgcctgacc tggagcttac tggtgtcact
     4621 tcctcaggcg ttcaagacca gcctgggcaa tatcgtgaga ctccatctct taaaaaaaaa
     4681 aaaaaaaagg attagccaag tgtggtggca tgaacctgtg gtcccagcta cttaagaggc
     4741 tgaggcagac agattgcttg agcctgggaa gtcgaagctg cagtgagcca tgatcatgcc
     4801 actgcactcc agcctgggtg acagagcaag accctgtctc aaaaaaataa aaatgaaaaa
     4861 aatctgtgtt cccaagttcc aagtgatgct gat
//
