LOCUS       sequence-348085-        7057 bp    DNA     circular SYN 15-APR-2025
DEFINITION  knockout mouse Gpi1.
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 Apr 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-Gpi1
FEATURES             Location/Qualifiers
     source          1..7057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     213..233
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     primer_bind     369..391
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     LTR             494..919
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     CDS             complement(1040..1840)
                     /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(1151..1170)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1761..1780
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(1835..1891)
                     /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             complement(1901..2386)
                     /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"
     promoter        complement(2406..2617)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_RNA        complement(2724..2799)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2821..3069)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2879..2898)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3049..3069)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(3116..3531)
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     complement(3132..3148)
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     complement(3164..3186)
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     misc_feature    3732..3931
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     LTR             3994..4169
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     promoter        complement(4170..4373)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4199..4219)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4374..4753
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(4853..4871)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4939..5043
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5044..5112,5113..5904)
                     /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     complement(5262..5281)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6075..6663
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6564..6583
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6817..6834
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(6915..6934)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        complement(join(6956..7057,1..228))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(6970..7057,1..48)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 gccatggggc ggagaatggg cggaactggg cggagttagg ggcgggatgg gcggagttag
       61 gggcgggact atggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg
      121 ggagcctggg gactttccac acctggttgc tgactaattg agatgcatgc tttgcatact
      181 tctgcctgct ggggagcctg gggactttcc acaccctaac tgacacacat tccacagccg
      241 gatctacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
      301 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
      361 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
      421 gcagccagct tacctcccgg tggtgggtcg gtggtccctg ggcaggggtc tcccgatccc
      481 ggacgagccc ccaaatgaaa gacccccgct gacgggtagt caatcactca gaggagaccc
      541 tcccaaggaa cagcgagacc acaagtcgga tgcaactgca agagggttta ttggatacac
      601 gggtacccgg gcgactcagt caatcggagg actggcgccc cgagtgaggg gttgtgggct
      661 cttttattga gctcggggag cagaagcgcg cgaacagaag cgagaagcga actgattggt
      721 tagttcaaat aaggcacagg gtcatttcag gtccttgggg caccctggaa acatctgatg
      781 gttctctagg gtcgaccctg ttccatctgt tcctgacctt gatctgaact tctctattct
      841 cagttatgta tttttccatg ccttgcaaaa tggcgttact taagctagct tgccaaacct
      901 acaggtgggg tctttcattc cccccttttt ctggagacta aataaaatct tttattttat
      961 ctatggctcg tactctatag gcttcagctg gtgatattgt tgagtcaaaa ctagagcctg
     1021 gaccactgat gatatctaat cagaagaact cgtcaagaag gcgatagaag gcgatgcgct
     1081 gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa
     1141 gctcttcagc aatatcacgg gtagccaacg ctatgtcctg atagcggtcc gccacaccca
     1201 gccggccaca gtcgatgaat ccagaaaagc ggccattttc caccatgata ttcggcaagc
     1261 aggcatcgcc atgggtcacg acgagatcct cgccgtcggg catgcgcgcc ttgagcctgg
     1321 cgaacagttc ggctggcgcg agcccctgat gctcttcgtc cagatcatcc tgatcgacaa
     1381 gaccggcttc catccgagta cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg
     1441 ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc atcagccatg atggatactt
     1501 tctcggcagg agcaaggtga gatgacagga gatcctgccc cggcacttcg cccaatagca
     1561 gccagtccct tcccgcttca gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg
     1621 tggccagcca cgatagccgc gctgcctcgt cctgcagttc attcagggca ccggacaggt
     1681 cggtcttgac aaaaagaacc gggcgcccct gcgctgacag ccggaacacg gcggcatcag
     1741 agcagccgat tgtctgttgt gcccagtcat agccgaatag cctctccacc caagcggccg
     1801 gagaacctgc gtgcaatcca tcttgttcaa tcatggggcc ggggttctcc tccacgtcgc
     1861 cggcctgctt cagcaggctg aagttggtgg cgccgctgcc cagagaaatg aagtccaggg
     1921 cttggaggag ggagagggaa agcagcagca gcagcatcca ggatgtgttc tgaaccagca
     1981 ggcttatgcc gccacacttg accagtttgt ctctatacac actgatactt ttattggagc
     2041 tcatgggatt cgcgcccgag actcgaagct cacaaaagta gtcgccctca tccttggtgg
     2101 tgaagttggc tagggtaagg accttgatat agggctggtt ggagagggtg acgcgggagc
     2161 ggtacgtgtg ctcgggtatc ccgagggtgc ctgagagcac gtgcttcctc ttctctcggg
     2221 tcaggctgaa ctcatgctgg atggagttat ccttggtgtt attctcatgg cggcagtcca
     2281 ggcgaaggtt ttggttcacc aggcaggctg tcaggctggt caccttctgc cctcgggaca
     2341 cctgcaagac tgagagcagg agagcgacgc tgatggctgg gttcatggtg gccgcgtcac
     2401 gacacctgtg ttctggcggc aaacccgttg cgaaaaagaa cgttcacggc gactactgca
     2461 cttatatacg gttctccccc accctcggga aaaaggcgga gccagtacac gacatcactt
     2521 tcccagttta ccccgcgcca ccttctctag gcaccggttc aattgccgac ccctcccccc
     2581 aacttctcgg ggactgtggg cgatgtgcgc tctgcccact gacgggcacc ggagccactc
     2641 gaggggccgg gagcatgcga cgtcgggccc aattcgccga attcccggcc cggcaccttg
     2701 tctcaagatc taaattcaaa aaagcaccga ctcggtgcca ctttttcaag ttgataacgg
     2761 actagcctta ttttaacttg ctatttctag ctctaaaacc cgatgccgat gttgatgatc
     2821 ggtgtttcgt cctttccaca agatatataa agccaagaaa tcgaaatact ttcaagttac
     2881 ggtaagcata tgatagtcca ttttaaaaca taattttaaa actgcaaact acccaagaaa
     2941 ttattacttt ctacgtcacg tattttgtac taatatcttt gtgtttacag tcaaattaat
     3001 tctaattatc tctctaacag ccttgtatcg tatatgcaaa tatgaaggaa tcatgggaaa
     3061 taggccctct ctagatctag aatcgataga tcgttaatta aagatcttca attccggcgc
     3121 ctagagaagg agtgagggct ggataaaggg aggatcgagg cggggtcgaa cgaggaggtt
     3181 caagggggag agacggggcg gatggaggaa gaggaggcgg aggcttaggg tgtacaaagg
     3241 gcttgaccca gggagggggg tcaaaagcca aggcttccca ggtcacgatg taggggacct
     3301 ggtctgggtg tccatgcggg ccaggtgaaa agaccttgat cttaacctgg gtgatgaggt
     3361 ctcggttaaa ggtgccgtct cgcggccatc cgacgttaaa ggttggccat tctgcagagc
     3421 agaaggtaac caacgtctct tcttgacatc taccgactgg ttgtgagcga tccgctcgac
     3481 atctttccag tgacctaagg tcaaacttaa gggagtggta acagtctggc cctaattttc
     3541 agacaaatac agaaacacag tcagacagag acaacacaga acgatgctgc agcgctgcag
     3601 cagacaagac gcgcggcttc ggttccaaac cgaaagcaaa aattcagacg gaggcgggaa
     3661 ctgttttagg ttctcgtctc ctaccagaac cacatatcct gacggggtcg gattccacat
     3721 cgactccctt cctcaggtcg ggccacaaaa acggccccca aagtccctgg gacgtctccc
     3781 agggttgcgg ccgggtgttc agaactcgtc agttccacca cgggtccgcc agatacagag
     3841 ctagttagct aactagtaca gacgcaggcg cataacatca aacatagaca ctagacaatc
     3901 ggacagacac agataagttg ctggccagct tacctcccgg tggtgggtcg gtggtccctg
     3961 ggcaggggtc tccaaatccc ggacgagccc ccaaatgaaa gacccccgtc gtgggtagtc
     4021 aatcactcag aggagaccct cccaaggaac agcgagacca cgattcggat gcaaacagca
     4081 agaggcttta ttgggaatac gggtacccgg gcgacgcagt ctatcggagg actggcgcgc
     4141 cgagtgaggg gttgtgggct cttttattga gctctgctta tatagacctc ccaccgtaca
     4201 cgcctaccgc ccatttgcgt caatggggcg gagttgttac gacattttgg aaagtcccgt
     4261 tgattttggt gccaaaacaa actcccattg acgtcaatgg ggtggagact tggaaatccc
     4321 cgtgagtcaa accgctatcc acgcccattg atgtactgcc aaaaccgcat caccatggta
     4381 atagcgatga ctaatacgta gatgtactgc caagtaggaa agtcccataa ggtcatgtac
     4441 tgggcataat gccaggcggg ccatttaccg tcattgacgt caataggggg cgtacttggc
     4501 atatgataca cttgatgtac tgccaagtgg gcagtttacc gtaaatactc cacccattga
     4561 cgtcaatgga aagtccctat tggcgttact atgggaacat acgtcattat tgacgtcaat
     4621 gggcgggggt cgttgggcgg tcagccaggc gggccattta ccgtaagtta tgtaacgcgg
     4681 aactccatat atgggctatg aactaatgac cccgtaattg attactatta ataactagtc
     4741 aataatcaat gtcaacatgg cggtaatgtt ggacatgagc caatataaat gtacatatta
     4801 tgatatggat acaacgtatg caatggccaa gctaattctt gaacacgaaa gggcctcgtg
     4861 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     4921 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     4981 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     5041 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     5101 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     5161 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     5221 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     5281 tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     5341 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     5401 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     5461 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     5521 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     5581 atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     5641 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     5701 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     5761 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     5821 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     5881 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     5941 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     6001 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     6061 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     6121 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     6181 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     6241 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     6301 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     6361 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     6421 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     6481 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     6541 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     6601 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     6661 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     6721 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     6781 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     6841 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     6901 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta agctttttgc
     6961 aaaagcctag gcctccaaaa aagcctcctc actacttctg gaatagctca gaggccgagg
     7021 cggcctcggc ctctgcataa ataaaaaaaa ttagtca
//
