LOCUS       sequence-466419-        7714 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  lentiviral overexpression vector for RAP2A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7714)
  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 7714)
  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-Rap2A-3xFLAG
FEATURES             Location/Qualifiers
     source          1..7714
                     /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             4025..4090
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    4138..4255
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4304..4814
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4733..4751
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4823..5218
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MVPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             5290..5523
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5595..5716
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5632..5651)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5686..5705
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5756..5891
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5911..5930)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5912..5930)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5940..5957)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5940..5956)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5949..5971)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6098..6553
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6185..6204)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6395..6416
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6579..6683
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6684..6752,6753..7544)
                     /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(6902..6921)
                     /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 taattaaagg gacgatgcgc gagtacaaag tggtggtgct
     3481 gggctcgggc ggggtaggca aatccgccct gaccgtgcag ttcgtgaccg gcaccttcat
     3541 cgagaaatac gaccccacca tcgaggactt ctaccgcaag gagatcgagg tggattcgtc
     3601 gccgtcggtg ctggagatcc tggacacggc gggcaccgag cagttcgcgt ccatgcggga
     3661 cctgtacatc aagaacggcc agggcttcat cctcgtctac agcctcgtca accagcagag
     3721 cttccaggac atcaagccca tgcgggacca gatcatccgc gtgaagcggt atgagaaagt
     3781 gccagtcatc ttggttggga acaaagtgga cctggaaagt gagagagaag tatcgtccag
     3841 cgaaggcaga gcccttgctg aagagtgggg ctgccccttt atggaaactt ccgctaagag
     3901 taaaacaatg gtggacgaac tctttgcaga aattgtgagg cagatgaact atgctgctca
     3961 gcctgacaaa gatgacccat gctgttctgc atgtaacata caaggaggca gcgcggccgc
     4021 aggcgactac aaagaccatg acggtgatta taaagatcat gacattgatt acaaggatga
     4081 cgatgacaag taataagaat tctcgacctc gagacaaatg gcagtattca tccacaattt
     4141 taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc
     4201 aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt
     4261 ttattacagg gacagcagag atccactttg gccgcggctc gagggggttg gggttgcgcc
     4321 ttttccaagg cagccctggg tttgcgcagg gacgcggctg ctctgggcgt ggttccggga
     4381 aacgcagcgg cgccgaccct gggtctcgca cattcttcac gtccgttcgc agcgtcaccc
     4441 ggatcttcgc cgctaccctt gtgggccccc cggcgacgct tcctgctccg cccctaagtc
     4501 gggaaggttc cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc gcacgtctca
     4561 ctagtaccct cgcagacgga cagcgccagg gagcaatggc agcgcgccga ccgcgatggg
     4621 ctgtggccaa tagcggctgc tcagcagggc gcgccgagag cagcggccgg gaaggggcgg
     4681 tgcgggaggc ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg cggtgttccg
     4741 cattctgcaa gcctccggag cgcacgtcgg cagtcggctc cctcgttgac cgaatcaccg
     4801 acctctctcc ccagggggat ccatggttcc tttgtctcaa gaagaatcca ccctcattga
     4861 aagagcaacg gctacaatca acagcatccc catctctgaa gactacagcg tcgccagcgc
     4921 agctctctct agcgacggcc gcatcttcac tggtgtcaat gtatatcatt ttactggggg
     4981 accttgtgca gaactcgtgg tgctgggcac tgctgctgct gcggcagctg gcaacctgac
     5041 ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg agcccctgcg gacggtgccg
     5101 acaggtgctt ctcgatctgc atcctgggat caaagccata gtgaaggaca gtgatggaca
     5161 gccgacggca gttgggattc gtgaattgct gccctctggt tatgtgtggg agggctaagg
     5221 tacctttaag accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa
     5281 aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt tttgcttgta
     5341 ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc
     5401 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt
     5461 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta
     5521 gcagtagtag ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat
     5581 cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     5641 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     5701 tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc
     5761 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     5821 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     5881 ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta cgcgcgctca
     5941 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     6001 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     6061 ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta
     6121 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     6181 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     6241 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     6301 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     6361 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     6421 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     6481 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     6541 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     6601 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     6661 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     6721 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     6781 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     6841 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     6901 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     6961 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     7021 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     7081 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     7141 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     7201 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     7261 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     7321 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     7381 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     7441 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     7501 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     7561 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     7621 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     7681 cgtcagaccc cgtagaaaag atcaaaggat cttc
//
