LOCUS       sequence-486775-        5869 bp    DNA     circular SYN 19-SEP-2025
DEFINITION  Lentiviral vector expressing mouse Pkm2-T328A mutant tagged with an 
            N-terminal EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5869)
  TITLE     Alpha-ketoglutarate mitigates insulin resistance and metabolic 
            inflexibility in Ataxia-Telangiectasia
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5869)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 19, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: FUGW-EGFP-mPkm2-T328A
FEATURES             Location/Qualifiers
     source          1..5869
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..36
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     21..37
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1704..1720)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1704..1720)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1749..1768)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1750..1768)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1778..1795)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1778..1794)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1787..1809)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(2006..2800)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(2006..2800)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2117..2136)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2727..2746
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(join(2809..3600,3601..3669))
                     /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     3432..3451
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3670..3774)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3844..4202
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3931..3950)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4141..4162
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4234..4267)
                     /label=lox71/66
                     /bound_moiety="Cre recombinase"
                     /note="loxP double mutant with a low affinity for Cre 
                     recombinase (Araki et al., 2010). 8-bp core sequence 
                     (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      4831..5419
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5320..5339
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5573..5590
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5707..5728
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5743..5748,5749..5766,5767..5773)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5781..5797
                     /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     5786..5808
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5805..5821
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5805..5821
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5840..5860
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5842..5860
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ctgggtaccg ggccccccct cgaggtcgac ggtatcgata agcttgatat ccactgtgga
       61 attcggccct ttcaaggtac aggcactaca cgcatggtgt tggtgaatcc agagccaggg
      121 cgccacccgg tcagcacaat gaccacatct cccttcttga agaagcctcg ggccttgcca
      181 acatccatgg ccaagtttac acgaaggtcg acatcctcag cccaggcatt cagcacggca
      241 tccttacaca gcacagggaa gatgccacgg tacagatggg cctggcgagc agtctgggga
      301 tttcgagtca cggcaatgat aggagcccga gggcggtacc tggccacttg gtgagcactc
      361 ctgccagact tggtgagcac gataatggcc ccactgcagc acttgaagga ggcctccacg
      421 gcacccacgg cggcagcttc tgtggggtcg ctggtaatgg gcgccaggcg gcggagttcc
      481 tcgaatagct gcaagtggta gatggcagcc tctgcctctc gggcaatcag gtgctgcatg
      541 cgaacagcct ccagagggta gtcccccttg gctgtttctc cagacagcat gatgcagtct
      601 gctccatcca ggactgcatt ggccacatca ctgccttcag cacgggtggg gcgtggcttc
      661 ttgatcatgc tctccagcat ctgggcggca cagatgacag gcttcccagc tcggttgcat
      721 cgcccgatca tcatcttctg agccaggaag accttctctg caggaatctc aatgcccagg
      781 tcaccacgag ccaccatgat cccatcactg gcctccaaga tctcatcaaa cctgcggacg
      841 ccttcatggt tctcgatttt gctgatgatc ttgatgttct tgcccttctc tcccagcacc
      901 ttcctgactt catgcacgtc ggctgccttg cggatgaaag atgcaaacac catgtccaca
      961 tcctgctcca ccccaaactt caggtcctgg atgtcctttt ccgacacagc ggggagatcc
     1021 acagcagcgc ccggcaggtt cacgcccttc ttgctgccca aggagccacc attctccacc
     1081 tccgtcacca ggaagtcagc gcctttctcc ttcacctgca gtgagatgag cccatcgtcc
     1141 acgtagatct tgctgcccac ctccaccacc ttgcagatgt tcttgtagtc cagccacagg
     1201 atgttctcgt cacacttctc catgtaagcg ttgtccaggg tgatcttcag agtggctccc
     1261 ttcttcagct ccacctcagc ggtgccgctg cccttgatga gtccagtccg gatctcaggt
     1321 ccctttgtat ccagagccac cgcaacagga cggtagagaa tgggatcaga tgcaaagctt
     1381 tctgtggctt cacggacatt cttgatggtc tctgcatggt actcatgggt tccatgagag
     1441 aaattcagcc gagccacatt cattccagac ttaatcatct ccttcagcat ctccacagat
     1501 cgggaagcag gcccaatggt acaaatgatg ccagtgttgc gggccgtgat gggggcagag
     1561 tcaatgtcca ggcggcacat gtgttccagg aaggtgtcag ccatggctgc atggagctgc
     1621 tgggtctgaa tgaaggcagt ccctgcttca ctgtgtggct tcggcataag ggcgaattcc
     1681 acattggtcg ctgcagcccg ggggatccac tagttctaga gcggccgcac cgcgggagct
     1741 ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc
     1801 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     1861 ccagctggcg taatagcgaa gaggcccgca ccgattaaat tttggtcatg agattatcaa
     1921 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     1981 tatatgagta aacttggtct gacagtcaga agaactcgtc aagaaggcga tagaaggcga
     2041 tgcgctgcga atcgggagcg gcgataccgt aaagcacgag gaagcggtca gcccattcgc
     2101 cgccaagttc ttcagcaata tcacgggtag ccaacgctat gtcctgatag cggtccgcca
     2161 cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc atgatattcg
     2221 gcaagcaggc atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg ctcgccttga
     2281 gcctggcgaa cagttcggct ggcgcgagcc cctgatgttc ttcgtccaga tcatcctgat
     2341 cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt
     2401 cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca gccatgatgg
     2461 atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc acttcgccca
     2521 atagcagcca gtcccttccc gcttcagtga caacgtcgag cacagctgcg caaggaacgc
     2581 ccgtcgtggc cagccacgat agccgcgctg cctcgtcttg cagttcattc agggcaccgg
     2641 acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg aacacggcgg
     2701 catcagagca gccgattgtc tgttgtgccc agtcatagcc gaatagcctc tccacccaag
     2761 cggccggaga acctgcgtgc aatccatctt gttcaatcat tagtgtcctt accaatgctt
     2821 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     2881 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     2941 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     3001 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     3061 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     3121 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     3181 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     3241 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     3301 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     3361 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     3421 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     3481 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     3541 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     3601 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     3661 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     3721 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     3781 tccccgaaaa gtgccacctt aatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     3841 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3901 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3961 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     4021 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     4081 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     4141 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     4201 tacagttaat taaagggaac aaaagctggc atgtaccgtt cgtatagcat acattatacg
     4261 aacggtacgc tccaattcgc cctttaatta actgttccaa ctttcaccat aatgaaataa
     4321 gatcactacc gggcgtattt tttgagttgt cgagattttc aggagctaag gaagctaaaa
     4381 tggagaaaaa aatcactgga tataccaccg agtactgcga tgagtggcag ggcggggcgt
     4441 aattttttta aggcagttat tggtgccctt aaacgcctgg ttgctacgcc tgaataagtg
     4501 ataataagcg gatgaatggc agaaattcga aagcaaattc gacccggtcg tcggttcagg
     4561 gcagggtcgt taaatagccg cttatgtcta ttgctggttt accggtttat tgactaccgg
     4621 aagcagtgtg accgtgtgct tctcaaatgc ctgaggccag tttgctcagg ctctccccgt
     4681 ggaggtaata attgacgata tgatcctttt tttctgatca aaaaggatct aggtgaagat
     4741 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     4801 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     4861 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     4921 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     4981 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     5041 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     5101 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     5161 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     5221 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     5281 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     5341 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     5401 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     5461 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     5521 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     5581 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     5641 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     5701 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgctccc
     5761 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
     5821 ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaag
//
