LOCUS       sequence-318844-        6448 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human TK1 with a N-terminal GFP-FLAG tag. Confers 
            Puromycin resistance. Inducible in T-REx cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6448)
  AUTHORS   Demange P, Joly E, Marcoux J, Zanon PRA, Listunov D, Rulliere P, 
            Barthes C, Noirot C, Izquierdo JB, Rozie A, Pradines K, Hee R, de 
            Brito MV, Marcellin M, Serre RF, Bouchez O, Burlet-Schiltz O, 
            Oliveira MCF, Ballereau S, Bernardes-Genisson V, Maraval V, Calsou 
            P, Hacker SM, Genisson Y, Chauvin R, Britton S
  TITLE     SDR enzymes oxidize specific lipidic alkynylcarbinols into cytotoxic
            protein-reactive species.
  JOURNAL   Elife. 2022 May 10;11. pii: 73913. doi: 10.7554/eLife.73913.
  PUBMED    35535493
REFERENCE   2  (bases 1 to 6448)
  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: "Elife."; 
            date: "2022-05-10"; volume: "11. pii: 73913. doi"; pages: " 
            10.7554/eLife.73913"
COMMENT     Sequence Label: pICE-EGFP-FLAG-TK1
FEATURES             Location/Qualifiers
     source          1..6448
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1..3,4..6,7..717)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1..3,4..6,7..717)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(46..67)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(307..326)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     654..675
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             733..756
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1507..1524)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1513..1737
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1783..2211
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1870..1889)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2080..2101
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2220..2240)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2225..2554
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2405..2540
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2467..2486
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2606..3205
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2606..2625)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3102..3122
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3335..3456
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3372..3391)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3426..3445
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3506..3522)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3506..3522)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3519..3541)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3530..3546
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3554..3560,3561..3578,3579..3584))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3599..3620
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3737..3754)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3908..4496)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3988..4007)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4667..5458,5459..5527))
                     /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     5290..5309
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5528..5632)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        5704..6083
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6084..6287
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6238..6258
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    6289..6307
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6310..6328
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     6338..6362
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      join(6443..6448,1..4)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
       61 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
      121 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      181 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
      241 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
      301 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
      361 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
      421 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
      481 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
      541 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
      601 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
      661 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc
      721 ggactcagat ctgattacaa ggatgacgac gataagctcg agagctgcat taacctgccc
      781 actgtgctgc ctggctcccc cagcaagacc cgggggcaga tccaggtgat tctcgggccg
      841 atgttctcag gaaaaagcac agagttgatg agacgcgtcc gtcgcttcca gattgctcag
      901 tacaagtgcc tggtgatcaa gtatgccaaa gacactcgct acagcagcag cttctgcaca
      961 catgaccgga acaccatgga ggcactgccc gcctgcctgc tccgagacgt ggcccaggag
     1021 gccctgggcg tggctgtcat aggcatcgac gaggggcagt ttttccctga catcgtggag
     1081 ttctgcgagg ccatggccaa cgccgggaag accgtaattg tggctgcact ggatgggacc
     1141 ttccagagga agccatttgg ggccatcctg aacctggtgc cgctggccga gagcgtggtg
     1201 aagctgacgg cggtgtgcat ggagtgcttc cgggaagccg cctataccaa gaggctcggc
     1261 acagagaagg aggtcgaggt gattggggga gcagacaagt accactccgt gtgtcggctc
     1321 tgctacttca agaaggcctc aggccagcct gccgggccgg acaacaaaga gaactgccca
     1381 gtgccaggaa agccagggga agccgtggct gccaggaagc tctttgcccc acagcagatt
     1441 ctgcaatgca gccctgccaa ctgagcggcc gcacgcgtct gcagtctaga taaacccgct
     1501 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     1561 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     1621 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     1681 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     1741 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     1801 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     1861 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     1921 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     1981 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     2041 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     2101 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     2161 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg
     2221 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     2281 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     2341 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     2401 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     2461 tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg
     2521 aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat
     2581 tttcggatct gatcagcacg tgaccatgac cgagtacaag cccacggtgc gcctcgccac
     2641 ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc
     2701 cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact
     2761 cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc
     2821 ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg
     2881 cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct
     2941 cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc
     3001 cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga
     3061 gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga
     3121 gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg
     3181 catgacccgc aagcccggtg cctgacacgt gctacgagat ttcgattcca ccgccgcctt
     3241 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     3301 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     3361 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     3421 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctag ccgtcgacct
     3481 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     3541 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     3601 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3661 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3721 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3781 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     3841 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     3901 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     3961 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4021 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4081 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4141 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4201 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4261 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4321 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     4381 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4441 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4501 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4561 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4621 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     4681 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     4741 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     4801 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     4861 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     4921 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     4981 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     5041 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     5101 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     5161 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     5221 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     5281 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     5341 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     5401 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     5461 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     5521 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5581 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5641 cccgaaaagt gccacctgac gtcgacggat cgggagatcc tgtacgggcc agatatccgc
     5701 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
     5761 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     5821 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     5881 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     5941 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     6001 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     6061 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
     6121 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     6181 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
     6241 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc cctatcagtg
     6301 atagagatct ccctatcagt gatagagatc gtcgacgagc tcgtttagtg aaccgtcaga
     6361 tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca
     6421 gcctccggca agcttaccgg tcgccacc
//
