LOCUS       sequence-429397-        7999 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral overexpression of human DHFR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7999)
  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 7999)
  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-DHFR-3xFLAG
FEATURES             Location/Qualifiers
     source          1..7999
                     /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             4037..4102
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    4150..4267
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4316..4826
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4745..4763
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4848..5447
                     /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(4848..4867)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5344..5364
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5575..5808
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5880..6001
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5917..5936)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5971..5990
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6041..6176
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6196..6215)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6197..6215)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6225..6242)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6225..6241)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6234..6256)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6383..6838
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6470..6489)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6680..6701
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6864..6968
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6969..7037,7038..7829)
                     /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(7187..7206)
                     /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 taattaatgc tgtcatggta ggatccttga attgtatcgt
     3481 cgcggtatct caaaatatgg ggataggaaa aaacggcgac ttgccgtggc cgccgcttag
     3541 aaacgagttc aggtactttc agcgaatgac taccacatct agcgtcgagg ggaagcagaa
     3601 ccttgtgatt atgggcaaaa aaacatggtt ttcaataccg gagaagaacc ggcccctcaa
     3661 aggccgcatt aatttggtat tgagtagaga actgaaagag ccaccccagg gtgctcattt
     3721 tctgagtagg tcccttgacg atgctctgaa gttgacagaa cagcctgagt tggcgaacaa
     3781 ggtggatatg gtatggatcg tcggaggctc atctgtatac aaagaagcca tgaaccatcc
     3841 cggccacctt aaactgtttg ttaccaggat aatgcaagac ttcgaatctg atacattttt
     3901 tcccgagatc gacttggaaa agtacaaact gttgcctgaa taccctggtg ttctctcaga
     3961 tgttcaggag gagaagggga taaaatataa gtttgaggtt tacgagaaga acgatggagg
     4021 cagcgcggcc gcaggcgact acaaagacca tgacggtgat tataaagatc atgacattga
     4081 ttacaaggat gacgatgaca agtaataaga attctcgacc tcgagacaaa tggcagtatt
     4141 catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
     4201 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
     4261 aaattttcgg gtttattaca gggacagcag agatccactt tggccgcggc tcgagggggt
     4321 tggggttgcg ccttttccaa ggcagccctg ggtttgcgca gggacgcggc tgctctgggc
     4381 gtggttccgg gaaacgcagc ggcgccgacc ctgggtctcg cacattcttc acgtccgttc
     4441 gcagcgtcac ccggatcttc gccgctaccc ttgtgggccc cccggcgacg cttcctgctc
     4501 cgcccctaag tcgggaaggt tccttgcggt tcgcggcgtg ccggacgtga caaacggaag
     4561 ccgcacgtct cactagtacc ctcgcagacg gacagcgcca gggagcaatg gcagcgcgcc
     4621 gaccgcgatg ggctgtggcc aatagcggct gctcagcagg gcgcgccgag agcagcggcc
     4681 gggaaggggc ggtgcgggag gcggggtgtg gggcggtagt gtgggccctg ttcctgcccg
     4741 cgcggtgttc cgcattctgc aagcctccgg agcgcacgtc ggcagtcggc tccctcgttg
     4801 accgaatcac cgacctctct ccccaggggg atccaccgga gcttaccatg accgagtaca
     4861 agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg
     4921 ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc
     4981 gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt
     5041 gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg
     5101 gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg
     5161 cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc
     5221 tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc
     5281 tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc
     5341 cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc
     5401 ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacgc ccgccccacg
     5461 acccgcagcg cccgaccgaa aggagcgcac gaccccatgc atcggtacct ttaagaccaa
     5521 tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag
     5581 ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt
     5641 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     5701 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     5761 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat
     5821 gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga
     5881 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     5941 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     6001 atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc
     6061 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     6121 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     6181 gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta
     6241 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     6301 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     6361 cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     6421 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     6481 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     6541 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     6601 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     6661 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     6721 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     6781 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     6841 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     6901 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     6961 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     7021 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     7081 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     7141 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     7201 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     7261 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     7321 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     7381 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     7441 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     7501 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     7561 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     7621 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     7681 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     7741 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     7801 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     7861 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     7921 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     7981 aaaagatcaa aggatcttc
//
