LOCUS       sequence-452489-        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: pML318_SEC23A-N-term_XTEN80-mEGFP-3xHA_pTMPLT
FEATURES             Location/Qualifiers
     source          1..3993
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(105..209)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     308..330
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     322..339
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     323..339
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             731..811
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             join(824..826,827..1537)
                     /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(866..887)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1127..1146)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1474..1495
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1547..1570
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2220..2236)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2220..2236)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2233..2255)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(2408..2996)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2488..2507)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(3113..3137)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(3237..3993,1..35,36..104))
                     /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     3860..3879
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
       61 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
      121 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
      181 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc agatacctga
      241 aacaaaaccc atcgtacggc caaggaagtc tccaataact gtgatccacc acaagcgcca
      301 gggttttccc agtcacgacg ttgtaaaacg acggccagtc atgcataatc cgcacgcatc
      361 tggaataagg aagtgccatt ccgcctgacc tggagcttac tggtgtcact tctgggatta
      421 caggcatacg ccaccatgcc tggctaattt tgtattttta gtagagacgg ggtttcttcg
      481 tgttggtcag gctggactcg aactcccgac ctcaggtgat ccgcctgtct cggcctccca
      541 aagtgctggg attaccggcg tgagccacgg ggcccagctg gtcctgattt ctaaggtgtt
      601 aatattgctg aatgagggga gaacattcat attttgaacg ttcttaagag gatcctggat
      661 tttaaaatat tttaaaattt tgatacagaa ataagaatca aactccacaa tgagtggtgg
      721 tacttccggg tacccatacg atgttccaga ttacgcttat ccgtatgacg tcccggacta
      781 tgcatacccc tacgatgtac ccgattacgc cgctggagct ggcgtgagca agggcgagga
      841 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
      901 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
      961 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     1021 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     1081 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     1141 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     1201 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     1261 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     1321 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     1381 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccaa
     1441 gctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     1501 cgccgggatc actctcggca tggacgagct gtacaagggt ggcggattgg aagttttgtt
     1561 tcaaggtcca ggatctaacg gcagcggagg gccgagctct ggcgcacccc caccaagtgg
     1621 agggtcacct gccgggtccc caacatctac tgaagaaggc accagcgaat ccgcaacgcc
     1681 cgagtcaggc cctggtacct ccacagaacc atctgaaggt agtgcgcctg gttccccagc
     1741 tggaagccct acttccaccg aagaaggcac gtcaaccgaa ccaagtgaag gatctgcccc
     1801 tgggaccagc actgaaccat ctgagggtgc agggagtgga agtacaacct atttagaatt
     1861 cattcaacaa aatgaagaac gagatggagt ccgatttagt tggaatgttt ggccatcaag
     1921 tcgactggaa gctacaagaa tggttgttcc tgtggcagcc ctgtttacac cactgaaaga
     1981 gaggcctgac ttaccaccta ttcaatatga acctgttctg tgtagtagga ccacttgccg
     2041 tgcagttttg aatcctttat ggtaagtaaa atatatagaa aaactaatgt ggcaatgtga
     2101 ttttaaaatg tcggggtttt tctgcatatt tttataaaaa tcccgtcaga agaccgtcag
     2161 tgaaggctag gtggaggctc agtgatgata agtctgcgat ggtggatgca tgtgtcatgg
     2221 tcatagctgt ttcctgtgtg aaattgttat ccgctcagag ggcacaatcc tattccgcgc
     2281 tatccgacaa tctccaagac attaggtgga gttcagttcg gcgtatggca tatgtcgctg
     2341 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     2401 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     2461 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     2521 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     2581 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     2641 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     2701 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     2761 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     2821 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     2881 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     2941 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     3001 atcctttgat cttttctacg gggtctgacg ctctattcaa caaagccgcc gtcccgtcaa
     3061 gtcagcgtaa atgggtaggg ggcttcaaat cgtcctcgtg ataccaattc ggagcctgct
     3121 tttttgtaca aacttgttga taatggcaat tcaaggatct tcacctagat ccttttaaat
     3181 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     3241 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     3301 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     3361 gctgcaatga taccgcgaga gccacgctca ccggctccag atttatcagc aataaaccag
     3421 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     3481 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     3541 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     3601 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     3661 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     3721 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     3781 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     3841 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     3901 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     3961 tcgatgtaac ccactcgtgc acccaactga tct
//
