LOCUS       sequence-466429-        7723 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  lentiviral overexpression vector for DHFR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7723)
  AUTHORS   Flickinger KM, Mellado Fritz CA, Huggler KS, Wade GM, Chang GR, Fox 
            KC, Simcox JA, Cantor JR
  TITLE     Cytosolic NADK is conditionally essential for folate-dependent 
            nucleotide synthesis.
  JOURNAL   Nat Metab. 2025 May 2. doi: 10.1038/s42255-025-01272-3.
  PUBMED    40316835
REFERENCE   2  (bases 1 to 7723)
  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: "Nat Metab. 
            2025 May 2. doi: 10.1038/s42255-025-01272-3."
COMMENT     Sequence Label: pLJC2-Blast-DHFR
FEATURES             Location/Qualifiers
     source          1..7723
                     /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             4034..4099
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    4147..4264
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4313..4823
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4742..4760
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4832..5227
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MVPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             5299..5532
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5604..5725
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5641..5660)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5695..5714
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5765..5900
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5920..5939)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5921..5939)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5949..5966)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5949..5965)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5958..5980)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6107..6562
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6194..6213)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6404..6425
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6588..6692
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6693..6761,6762..7553)
                     /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(6911..6930)
                     /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 acgattaata
     4021 agcggccgca ggcgactaca aagaccatga cggtgattat aaagatcatg acattgatta
     4081 caaggatgac gatgacaagt aataagaatt ctcgacctcg agacaaatgg cagtattcat
     4141 ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
     4201 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa
     4261 ttttcgggtt tattacaggg acagcagaga tccactttgg ccgcggctcg agggggttgg
     4321 ggttgcgcct tttccaaggc agccctgggt ttgcgcaggg acgcggctgc tctgggcgtg
     4381 gttccgggaa acgcagcggc gccgaccctg ggtctcgcac attcttcacg tccgttcgca
     4441 gcgtcacccg gatcttcgcc gctacccttg tgggcccccc ggcgacgctt cctgctccgc
     4501 ccctaagtcg ggaaggttcc ttgcggttcg cggcgtgccg gacgtgacaa acggaagccg
     4561 cacgtctcac tagtaccctc gcagacggac agcgccaggg agcaatggca gcgcgccgac
     4621 cgcgatgggc tgtggccaat agcggctgct cagcagggcg cgccgagagc agcggccggg
     4681 aaggggcggt gcgggaggcg gggtgtgggg cggtagtgtg ggccctgttc ctgcccgcgc
     4741 ggtgttccgc attctgcaag cctccggagc gcacgtcggc agtcggctcc ctcgttgacc
     4801 gaatcaccga cctctctccc cagggggatc catggttcct ttgtctcaag aagaatccac
     4861 cctcattgaa agagcaacgg ctacaatcaa cagcatcccc atctctgaag actacagcgt
     4921 cgccagcgca gctctctcta gcgacggccg catcttcact ggtgtcaatg tatatcattt
     4981 tactggggga ccttgtgcag aactcgtggt gctgggcact gctgctgctg cggcagctgg
     5041 caacctgact tgtatcgtcg cgatcggaaa tgagaacagg ggcatcttga gcccctgcgg
     5101 acggtgccga caggtgcttc tcgatctgca tcctgggatc aaagccatag tgaaggacag
     5161 tgatggacag ccgacggcag ttgggattcg tgaattgctg ccctctggtt atgtgtggga
     5221 gggctaaggt acctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt
     5281 taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa gatctgcttt
     5341 ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
     5401 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
     5461 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
     5521 aaatctctag cagtagtagt tcatgtcatc ttattattca gtatttataa cttgcaaaga
     5581 aatgaatatc agagagtgag aggaacttgt ttattgcagc ttataatggt tacaaataaa
     5641 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     5701 tgtccaaact catcaatgta tcttatcatg tctggctcta gctatcccgc ccctaactcc
     5761 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     5821 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     5881 aggaggcttt tttggaggcc tagggacgta cccaattcgc cctatagtga gtcgtattac
     5941 gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     6001 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     6061 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc
     6121 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     6181 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     6241 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     6301 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     6361 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     6421 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     6481 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     6541 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     6601 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     6661 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     6721 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     6781 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     6841 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     6901 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     6961 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     7021 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     7081 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     7141 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     7201 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     7261 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     7321 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     7381 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     7441 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     7501 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     7561 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     7621 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     7681 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttc
//
