LOCUS       sequence-466418-        7726 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 7726)
  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 7726)
  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-3xFLAG
FEATURES             Location/Qualifiers
     source          1..7726
                     /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             4835..5230
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MVPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             5302..5535
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5607..5728
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5644..5663)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5698..5717
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5768..5903
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5923..5942)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5924..5942)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5952..5969)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5952..5968)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5961..5983)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6110..6565
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6197..6216)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6407..6428
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6591..6695
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6696..6764,6765..7556)
                     /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(6914..6933)
                     /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 atccatggtt cctttgtctc aagaagaatc
     4861 caccctcatt gaaagagcaa cggctacaat caacagcatc cccatctctg aagactacag
     4921 cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc actggtgtca atgtatatca
     4981 ttttactggg ggaccttgtg cagaactcgt ggtgctgggc actgctgctg ctgcggcagc
     5041 tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac aggggcatct tgagcccctg
     5101 cggacggtgc cgacaggtgc ttctcgatct gcatcctggg atcaaagcca tagtgaagga
     5161 cagtgatgga cagccgacgg cagttgggat tcgtgaattg ctgccctctg gttatgtgtg
     5221 ggagggctaa ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact
     5281 ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga caagatctgc
     5341 tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag ctctctggct
     5401 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt
     5461 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt
     5521 ggaaaatctc tagcagtagt agttcatgtc atcttattat tcagtattta taacttgcaa
     5581 agaaatgaat atcagagagt gagaggaact tgtttattgc agcttataat ggttacaaat
     5641 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     5701 gtttgtccaa actcatcaat gtatcttatc atgtctggct ctagctatcc cgcccctaac
     5761 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     5821 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     5881 gtgaggaggc ttttttggag gcctagggac gtacccaatt cgccctatag tgagtcgtat
     5941 tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     6001 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     6061 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt
     6121 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     6181 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     6241 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     6301 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     6361 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     6421 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     6481 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     6541 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
     6601 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     6661 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     6721 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     6781 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     6841 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     6901 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     6961 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     7021 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     7081 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     7141 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     7201 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     7261 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     7321 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     7381 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     7441 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     7501 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     7561 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     7621 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     7681 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttc
//
