LOCUS       sequence-348091-        7057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  knockout mouse Gapdh.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7057)
  AUTHORS   Wu L, Hollinshead KER, Hao Y, Au C, Kroehling L, Ng C, Lin WY, Li D,
            Silva HM, Shin J, Lafaille JJ, Possemato R, Pacold ME, 
            Papagiannakopoulos T, Kimmelman AC, Satija R, Littman DR
  TITLE     Niche-Selective Inhibition of Pathogenic Th17 Cells by Targeting 
            Metabolic Redundancy.
  JOURNAL   Cell. 2020 Aug 6;182(3):641-654.e20. doi: 
            10.1016/j.cell.2020.06.014. Epub 2020 Jul 1.
  PUBMED    32615085
REFERENCE   2  (bases 1 to 7057)
  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"; doi: 
            "10.1016/j.cell.2020.06.014"; journalName: "Cell"; date: "2020-08-6-
            6"; volume: "182"; issue: "3"; pages: "641-654.e20"
COMMENT     Sequence Label: pSIN-Gapdh
FEATURES             Location/Qualifiers
     source          1..7057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(60..77)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(231..819)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(311..330)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(990..1781,1782..1850))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1613..1632
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1851..1955)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2023..2041
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        2141..2520
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2521..2724
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2675..2695
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             2725..2900
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    2963..3162
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             3363..3778
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     3708..3730
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     3746..3762
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        3825..4073
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3825..3845
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3996..4015
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4095..4170
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4277..4488
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             4508..4993
                     /codon_start=1
                     /product="mouse GPI-anchored cell surface glycoprotein of 
                     the immunoglobulin superfamily"
                     /label=Thy1.1
                     /note="This allele of Thy1 is also known as CD90.1."
                     /translation="MNPAISVALLLSVLQVSRGQKVTSLTACLVNQNLRLDCRHENNTK
                     DNSIQHEFSLTREKRKHVLSGTLGIPEHTYRSRVTLSNQPYIKVLTLANFTTKDEGDYF
                     CELRVSGANPMSSNKSISVYRDKLVKCGGISLLVQNTSWMLLLLLSLSLLQALDFISL"
     CDS             5003..5059
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             5054..5854
                     /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="GPMIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQG
                     RPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEE
                     HQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQD
                     IALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5114..5133)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5724..5743
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     LTR             5975..6400
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(6503..6525)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6661..6681)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6666..6995
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6846..6981
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7017..7036
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc
       61 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
      121 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
      181 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
      241 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
      301 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
      361 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
      421 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
      481 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
      541 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
      601 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
      661 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
      721 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
      781 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
      841 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
      901 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
      961 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     1021 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     1081 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     1141 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     1201 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     1261 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctgcagg catcgtggtg
     1321 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     1381 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     1441 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     1501 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     1561 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg ggataatacc
     1621 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     1681 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     1741 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     1801 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     1861 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     1921 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     1981 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     2041 ccctttcgtg ttcaagaatt agcttggcca ttgcatacgt tgtatccata tcataatatg
     2101 tacatttata ttggctcatg tccaacatta ccgccatgtt gacattgatt attgactagt
     2161 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
     2221 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
     2281 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
     2341 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
     2401 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
     2461 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
     2521 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
     2581 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
     2641 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
     2701 tgggaggtct atataagcag agctcaataa aagagcccac aacccctcac tcggcgcgcc
     2761 agtcctccga tagactgcgt cgcccgggta cccgtattcc caataaagcc tcttgctgtt
     2821 tgcatccgaa tcgtggtctc gctgttcctt gggagggtct cctctgagtg attgactacc
     2881 cacgacgggg gtctttcatt tgggggctcg tccgggattt ggagacccct gcccagggac
     2941 caccgaccca ccaccgggag gtaagctggc cagcaactta tctgtgtctg tccgattgtc
     3001 tagtgtctat gtttgatgtt atgcgcctgc gtctgtacta gttagctaac tagctctgta
     3061 tctggcggac ccgtggtgga actgacgagt tctgaacacc cggccgcaac cctgggagac
     3121 gtcccaggga ctttgggggc cgtttttgtg gcccgacctg aggaagggag tcgatgtgga
     3181 atccgacccc gtcaggatat gtggttctgg taggagacga gaacctaaaa cagttcccgc
     3241 ctccgtctga atttttgctt tcggtttgga accgaagccg cgcgtcttgt ctgctgcagc
     3301 gctgcagcat cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaatt
     3361 agggccagac tgttaccact cccttaagtt tgaccttagg tcactggaaa gatgtcgagc
     3421 ggatcgctca caaccagtcg gtagatgtca agaagagacg ttggttacct tctgctctgc
     3481 agaatggcca acctttaacg tcggatggcc gcgagacggc acctttaacc gagacctcat
     3541 cacccaggtt aagatcaagg tcttttcacc tggcccgcat ggacacccag accaggtccc
     3601 ctacatcgtg acctgggaag ccttggcttt tgacccccct ccctgggtca agccctttgt
     3661 acaccctaag cctccgcctc ctcttcctcc atccgccccg tctctccccc ttgaacctcc
     3721 tcgttcgacc ccgcctcgat cctcccttta tccagccctc actccttctc taggcgccgg
     3781 aattgaagat ctttaattaa cgatctatcg attctagatc tagagagggc ctatttccca
     3841 tgattccttc atatttgcat atacgataca aggctgttag agagataatt agaattaatt
     3901 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg
     3961 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt
     4021 gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac accgacttac
     4081 cccggccttc tccagtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
     4141 caacttgaaa aagtggcacc gagtcggtgc ttttttgaat ttagatcttg agacaaggtg
     4201 ccgggccggg aattcggcga attgggcccg acgtcgcatg ctcccggccc ctcgagtggc
     4261 tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga
     4321 ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat
     4381 gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta
     4441 gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt gtcgtgacgc
     4501 ggccaccatg aacccagcca tcagcgtcgc tctcctgctc tcagtcttgc aggtgtcccg
     4561 agggcagaag gtgaccagcc tgacagcctg cctggtgaac caaaaccttc gcctggactg
     4621 ccgccatgag aataacacca aggataactc catccagcat gagttcagcc tgacccgaga
     4681 gaagaggaag cacgtgctct caggcaccct cgggataccc gagcacacgt accgctcccg
     4741 cgtcaccctc tccaaccagc cctatatcaa ggtccttacc ctagccaact tcaccaccaa
     4801 ggatgagggc gactactttt gtgagcttcg agtctcgggc gcgaatccca tgagctccaa
     4861 taaaagtatc agtgtgtata gagacaaact ggtcaagtgt ggcggcataa gcctgctggt
     4921 tcagaacaca tcctggatgc tgctgctgct gctttccctc tccctcctcc aagccctgga
     4981 cttcatttct ctgggcagcg gcgccaccaa cttcagcctg ctgaagcagg ccggcgacgt
     5041 ggaggagaac cccggcccca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     5101 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     5161 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     5221 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     5281 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     5341 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     5401 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     5461 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     5521 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     5581 caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     5641 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     5701 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     5761 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     5821 cgccttctat cgccttcttg acgagttctt ctgattagat atcatcagtg gtccaggctc
     5881 tagttttgac tcaacaatat caccagctga agcctataga gtacgagcca tagataaaat
     5941 aaaagatttt atttagtctc cagaaaaagg ggggaatgaa agaccccacc tgtaggtttg
     6001 gcaagctagc ttaagtaacg ccattttgca aggcatggaa aaatacataa ctgagaatag
     6061 agaagttcag atcaaggtca ggaacagatg gaacagggtc gaccctagag aaccatcaga
     6121 tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc
     6181 agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa aagagcccac
     6241 aacccctcac tcggggcgcc agtcctccga ttgactgagt cgcccgggta cccgtgtatc
     6301 caataaaccc tcttgcagtt gcatccgact tgtggtctcg ctgttccttg ggagggtctc
     6361 ctctgagtga ttgactaccc gtcagcgggg gtctttcatt tgggggctcg tccgggatcg
     6421 ggagacccct gcccagggac caccgaccca ccaccgggag gtaagctggc tgcctcgcgc
     6481 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     6541 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     6601 ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtag atccggctgt
     6661 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     6721 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     6781 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     6841 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     6901 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     6961 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct tactggctta actatgcggc
     7021 atcagagcag attgtactga gagtgcacca tatgcgg
//
