LOCUS       sequence-429393-        8233 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral overexpression of human TK2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8233)
  AUTHORS   Flickinger KM, Wilson KM, Rossiter NJ, Hunger AL, Vishwasrao PV, Lee
            TD, Mellado Fritz CA, Richards RM, Hall MD, Cantor JR
  TITLE     Conditional lethality profiling reveals anticancer mechanisms of 
            action and drug-nutrient interactions.
  JOURNAL   Sci Adv. 2024 Oct 4;10(40):eadq3591. doi: 10.1126/sciadv.adq3591. 
            Epub 2024 Oct 4.
  PUBMED    39365851
REFERENCE   2  (bases 1 to 8233)
  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: "Sci Adv."; 
            date: "2024-10-4"; volume: "10(40):eadq3591. doi"; pages: " 
            10.1126/sciadv.adq3591. Epub 2024 Oct 4"
COMMENT     Sequence Label: pLJC2-TK2-3xFLAG
FEATURES             Location/Qualifiers
     source          1..8233
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1010..1030
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1012..1030
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1058..1284
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1285..1465
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1512..1637
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2130..2363
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2548..2592
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     primer_bind     2890..2910
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        3199..3402
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3353..3373
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             4271..4336
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    4384..4501
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4550..5060
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4979..4997
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             5082..5681
                     /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(5082..5101)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5578..5598
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5809..6042
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    6114..6235
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6151..6170)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6205..6224
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6275..6410
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6430..6449)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6431..6449)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6459..6476)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6459..6475)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6468..6490)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6617..7072
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6704..6723)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6914..6935
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7098..7202
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7203..7271,7272..8063)
                     /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     complement(7421..7440)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
     1021 tcactaaagg gaacaaaagc tggagctgca agcttaatgt agtcttatgc aatactcttg
     1081 tagtcttgca acatggtaac gatgagttag caacatgcct tacaaggaga gaaaaagcac
     1141 cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg ccttattagg aaggcaacag
     1201 acgggtctga catggattgg acgaaccact gaattgccgc attgcagaga tattgtattt
     1261 aagtgcctag ctcgatacat aaacgggtct ctctggttag accagatctg agcctgggag
     1321 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     1381 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     1441 tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga
     1501 aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg
     1561 aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag
     1621 atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc
     1681 ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg
     1741 agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa
     1801 tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata
     1861 atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag
     1921 ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcagcag
     1981 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
     2041 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
     2101 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
     2161 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
     2221 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
     2281 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
     2341 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
     2401 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
     2461 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
     2521 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
     2581 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
     2641 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
     2701 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg
     2761 ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc
     2821 attcgattag tgaacggatc tcgacggtat cgatcacgag actagcctcg agcagcagcc
     2881 cccttcaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     2941 gttagagaga taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg
     3001 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     3061 gactatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     3121 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
     3181 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
     3241 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
     3301 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
     3361 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag
     3421 atccgctagc gctaccggtt taattaaccc ggctatgttg ctgtggccct tgcgagggtg
     3481 ggcagctaga gccctcagat gtttcggtcc gggttcacga gggagtccgg catcaggacc
     3541 cggacctcgg cgcgtacaac gacgcgcttg gccgccagat aaggagcaag agaaggaaaa
     3601 gaaatcagta atctgtgtgg agggtaacat agctagtggg aagacaacct gtctcgagtt
     3661 cttcagcaat gccacggacg tagaagtcct gacggaaccc gtatcaaaat ggaggaacgt
     3721 tcggggtcac aaccccctgg gtctcatgta ccatgatgca agcagatggg gtcttaccct
     3781 gcagacttat gtgcagctta ctatgttgga tcgacatacc cgcccgcagg tttctagtgt
     3841 acggttgatg gagaggagca tacacagtgc ccgctatatc tttgtcgaaa acctctacag
     3901 gtccggcaaa atgcccgagg tcgattacgt tgtactcagt gagtggtttg attggatact
     3961 gagaaatatg gacgtgtccg ttgacctcat cgtgtacctc cggactaatc ccgaaacctg
     4021 ctatcaacga ctcaagaagc gctgtaggga agaagagaag gtgatcccac tggagtatct
     4081 tgaagccatt caccatttgc acgaggaatg gttgatcaag ggttctctct ttcctatggc
     4141 agcaccggtg ttggtgatag aagccgacca tcacatggaa agaatgttgg agttgtttga
     4201 acagaacaga gacaggatac ttacaccaga aaatcgcaaa cactgcccag gaggcagcgc
     4261 ggccgcaggc gactacaaag accatgacgg tgattataaa gatcatgaca ttgattacaa
     4321 ggatgacgat gacaagtaat aagaattctc gacctcgaga caaatggcag tattcatcca
     4381 caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
     4441 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt
     4501 tcgggtttat tacagggaca gcagagatcc actttggccg cggctcgagg gggttggggt
     4561 tgcgcctttt ccaaggcagc cctgggtttg cgcagggacg cggctgctct gggcgtggtt
     4621 ccgggaaacg cagcggcgcc gaccctgggt ctcgcacatt cttcacgtcc gttcgcagcg
     4681 tcacccggat cttcgccgct acccttgtgg gccccccggc gacgcttcct gctccgcccc
     4741 taagtcggga aggttccttg cggttcgcgg cgtgccggac gtgacaaacg gaagccgcac
     4801 gtctcactag taccctcgca gacggacagc gccagggagc aatggcagcg cgccgaccgc
     4861 gatgggctgt ggccaatagc ggctgctcag cagggcgcgc cgagagcagc ggccgggaag
     4921 gggcggtgcg ggaggcgggg tgtggggcgg tagtgtgggc cctgttcctg cccgcgcggt
     4981 gttccgcatt ctgcaagcct ccggagcgca cgtcggcagt cggctccctc gttgaccgaa
     5041 tcaccgacct ctctccccag ggggatccac cggagcttac catgaccgag tacaagccca
     5101 cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt
     5161 tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca
     5221 ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg
     5281 cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg
     5341 tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc
     5401 aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca
     5461 ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg
     5521 gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca
     5581 acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag
     5641 gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg acgcccgccc cacgacccgc
     5701 agcgcccgac cgaaaggagc gcacgacccc atgcatcggt acctttaaga ccaatgactt
     5761 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa
     5821 ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc
     5881 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     5941 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     6001 gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt tcatgtcatc
     6061 ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt
     6121 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     6181 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     6241 tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     6301 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     6361 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta
     6421 cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt
     6481 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     6541 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     6601 ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     6661 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     6721 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     6781 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     6841 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     6901 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     6961 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     7021 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     7081 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     7141 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     7201 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     7261 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     7321 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     7381 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     7441 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     7501 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     7561 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     7621 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     7681 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     7741 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     7801 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     7861 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     7921 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     7981 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     8041 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     8101 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     8161 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     8221 tcaaaggatc ttc
//
