LOCUS       sequence-437198-        3363 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry clone with codon-optimized ORF sequence for subcloning
            into custom expression vectors.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3363)
  AUTHORS   Superti-Furga G, Lackner D, Wiedmer T, Ingles-Prieto A, Barbosa B, 
            Girardi E, Goldmann U, Gurtl B, Klavins K, Klimek C, Lindinger S, 
            Lineiro-Retes E, Muller AC, Onstein S, Redinger G, Reil D, Sedlyarov
            V, Wolf G, Crawford M, Everley R, ... Steppan CM
  TITLE     The RESOLUTE consortium: unlocking SLC transporters for drug 
            discovery.
  JOURNAL   Nat Rev Drug Discov. 2020 Jul;19(7):429-430. doi: 
            10.1038/d41573-020-00056-6.
  PUBMED    32265506
REFERENCE   2  (bases 1 to 3363)
  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"; doi: "10"; journalName: 
            "Nat Rev Drug Discov"; date: "2020-07"; volume: "19"; issue: "7"; 
            pages: "429-430"
COMMENT     Sequence Label: pDONR221-eGFP_mito
FEATURES             Location/Qualifiers
     source          1..3363
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     94..111
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(270..297)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(389..475)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     524..546
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     538..555
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     539..555
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    571..670
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             676..750
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             join(775..777,778..1488)
                     /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(817..838)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1078..1097)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1425..1446
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1490..1589)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1606..1625)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1607..1625)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1630..1646)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1630..1646)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1748..1767)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1759..2568
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1826..1845)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2715..3303
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3204..3223
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
       61 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
      121 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
      181 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tacgcgtacc
      241 gctagccagg aagagtttgt agaaacgcaa aaaggccatc cgtcaggatg gccttctgct
      301 tagtttgatg cctggcagtt tatggcgggc gtcctgcccg ccaccctccg ggccgttgct
      361 tcacaacgtt caaatccgct cccggcggat ttgtcctact caggagagcg ttcaccgaca
      421 aacaacagat aaaacgaaag gcccagtctt ccgactgagc ctttcgtttt atttgatgcc
      481 tggcagttcc ctactctcgc gttaacgcta gcatggatgt tttcccagtc acgacgttgt
      541 aaaacgacgg ccagtcttaa gctcgggccc caaataatga ttttattttg actgatagtg
      601 acctgttcgt tgcaacaaat tgatgagcaa tgctttttta taatgccaac tttgtacaaa
      661 aaagcaggct ccaccatgtc cgtcctgacg ccgctgctgc tgcggggctt gacaggctcg
      721 gcccggcggc tcccagtgcc gcgcgccaag atccattcgt tgctgcaggt cgacgtgagc
      781 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
      841 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
      901 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
      961 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     1021 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     1081 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     1141 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     1201 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     1261 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     1321 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     1381 acccagtcca agctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     1441 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagga cccagctttc
     1501 ttgtacaaag ttggcattat aagaaagcat tgcttatcaa tttgttgcaa cgaacaggtc
     1561 actatcagtc aaaataaaat cattatttgc catccagctg atatccccta tagtgagtcg
     1621 tattacatgg tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt
     1681 tacattgcac aagataaaaa tatatcatca tgaacaataa aactgtctgc ttacataaac
     1741 agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcgaggcc gcgattaaat
     1801 tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca
     1861 ggtgcgacaa tctatcgctt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat
     1921 ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg
     1981 gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta
     2041 ctcaccactg cgatccccgg aaaaacagca ttccaggtat tagaagaata tcctgattca
     2101 ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt
     2161 tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg
     2221 aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa
     2281 caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat
     2341 ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat
     2401 gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc
     2461 ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct
     2521 gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt
     2581 aattggttgt aacactggca gagcattacg ctgacttgac gggacggcgc aagctcatga
     2641 ccaaaatccc ttaacgtgag ttacgcgtcg ttccactgag cgtcagaccc cgtagaaaag
     2701 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     2761 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     2821 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     2881 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     2941 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     3001 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     3061 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3121 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     3181 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     3241 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     3301 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     3361 atg
//
