LOCUS       sequence-466417-        8677 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  lentiviral overexpression vector for SHMT2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8677)
  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 8677)
  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-SHMT2-3xFLAG
FEATURES             Location/Qualifiers
     source          1..8677
                     /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             4988..5053
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    5101..5218
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5267..5777
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     5696..5714
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             5786..6181
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MVPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             6253..6486
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    6558..6679
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6595..6614)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6649..6668
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6719..6854
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6874..6893)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6875..6893)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6903..6920)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6903..6919)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6912..6934)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7061..7516
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7148..7167)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7358..7379
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7542..7646
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7647..7715,7716..8507)
                     /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(7865..7884)
                     /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 taattaaagt tgcgatgctg tatttcagcc tcttctgggc
     3481 agcgagacca ctccagaggt gcggacaact ggtgcgaatg gcgattcgcg cacagcactc
     3541 taatgctgct caaactcaga caggcgaagc caacagaggt tggaccgggc aagagagttt
     3601 gtccgacagc gacccggaga tgtgggagtt gttgcagcga gagaaagatc gccaatgtcg
     3661 cggtctggaa ttgatagcga gtgagaattt ctgtagcaga gcagccttgg aggcgcttgg
     3721 atcctgtctg aacaataaat actcagaagg gtatccgggg aagcgctact atggaggggc
     3781 ggaggtagta gacgaaatcg agctgctgtg ccagcgccgc gctctcgaag cgttcgacct
     3841 ggatcccgct cagtggggcg ttaatgtaca accctattcc ggcagcccag ccaacctcgc
     3901 tgtctacaca gcactgctgc agcctcatga taggatcatg ggactggacc tcccagatgg
     3961 gggtcatctc acacatgggt acatgagcga tgttaaacga atatcagcaa cgtcaatatt
     4021 cttcgagagt atgccttata agcttaatcc caagactgga cttattgatt ataatcaatt
     4081 ggcactcacc gccaggttgt tccgcccgcg cctgataata gcagggactt cagcttacgc
     4141 caggctcata gactatgcca ggatgagaga agtttgtgat gaggttaaag cccatttgct
     4201 tgcagatatg gcccatatct cagggctggt ggccgcaaaa gttattccct cacccttcaa
     4261 gcacgccgac atcgtgacaa cgactaccca caagacgttg agaggggcaa ggtctggcct
     4321 cattttttac agaaaaggcg taaaggcggt cgatcccaaa acagggcggg agatccccta
     4381 tacatttgaa gaccgcatta attttgcggt atttcccagt cttcaagggg gccctcataa
     4441 ccacgcaata gcggccgtgg ctgtcgcact gaagcaggct tgtacgccaa tgtttaggga
     4501 gtactccctc caggtcctta agaatgcaag agctatggca gacgctcttc tggaaagagg
     4561 gtactctctg gtatctggtg gtacagataa ccatctggtg ctcgtagacc tgcgccctaa
     4621 gggtttggat ggagcgcggg ctgagagggt gttggagctt gttagcatta ccgcaaataa
     4681 gaatacgtgt cctggagacc gctccgctat cactcccgga gggttgcggt tgggggcgcc
     4741 cgctttgacc tcacgccagt tcagggagga cgactttagg agagtcgtgg attttataga
     4801 tgaaggagta aacattggat tggaggtaaa aagcaagact gcaaaactcc aagactttaa
     4861 aagtttcctt ctgaaggata gcgagacctc acaaagattg gctaacctgc gccagcgggt
     4921 cgaacagttc gcaagagcgt tcccaatgcc gggattcgat gagcatggag gaagtgcggc
     4981 cgcaggcgac tacaaagacc atgacggtga ttataaagat catgacattg attacaagga
     5041 tgacgatgac aagtaataag aattctcgac ctcgagacaa atggcagtat tcatccacaa
     5101 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat
     5161 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg
     5221 ggtttattac agggacagca gagatccact ttggccgcgg ctcgaggggg ttggggttgc
     5281 gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg cgtggttccg
     5341 ggaaacgcag cggcgccgac cctgggtctc gcacattctt cacgtccgtt cgcagcgtca
     5401 cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct ccgcccctaa
     5461 gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa gccgcacgtc
     5521 tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc cgaccgcgat
     5581 gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc cgggaagggg
     5641 cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc gcgcggtgtt
     5701 ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt gaccgaatca
     5761 ccgacctctc tccccagggg gatccatggt tcctttgtct caagaagaat ccaccctcat
     5821 tgaaagagca acggctacaa tcaacagcat ccccatctct gaagactaca gcgtcgccag
     5881 cgcagctctc tctagcgacg gccgcatctt cactggtgtc aatgtatatc attttactgg
     5941 gggaccttgt gcagaactcg tggtgctggg cactgctgct gctgcggcag ctggcaacct
     6001 gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct gcggacggtg
     6061 ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc atagtgaagg acagtgatgg
     6121 acagccgacg gcagttggga ttcgtgaatt gctgccctct ggttatgtgt gggagggcta
     6181 aggtaccttt aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag
     6241 aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt
     6301 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
     6361 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     6421 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     6481 ctagcagtag tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa
     6541 tatcagagag tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     6601 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     6661 aactcatcaa tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccat
     6721 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     6781 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     6841 cttttttgga ggcctaggga cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc
     6901 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     6961 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     7021 cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca
     7081 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     7141 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     7201 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     7261 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     7321 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     7381 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     7441 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     7501 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     7561 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     7621 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     7681 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     7741 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     7801 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     7861 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     7921 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     7981 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     8041 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     8101 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     8161 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     8221 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     8281 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     8341 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     8401 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     8461 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     8521 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     8581 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     8641 gagcgtcaga ccccgtagaa aagatcaaag gatcttc
//
