LOCUS       sequence-364045-        7057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  targeting mouse Ski gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7057)
  AUTHORS   Xu H, Wu L, Nguyen HH, Mesa KR, Raghavan V, Episkopou V, Littman DR
  TITLE     Arkadia-SKI/SnoN signaling differentially regulates TGF-beta-induced
            iTreg and Th17 cell differentiation.
  JOURNAL   J Exp Med. 2021 Nov 1;218(11). pii: 212614. doi: 
            10.1084/jem.20210777. Epub 2021 Sep 2.
  PUBMED    34473197
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"; journalName: "J Exp 
            Med."; date: "2021-11-1"; volume: "218(11). pii: 212614. doi"; 
            pages: " 10.1084/jem.20210777. Epub 2021 Sep 2"
COMMENT     Sequence Label: pSIN-Ski-gRNA1
FEATURES             Location/Qualifiers
     source          1..7057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     305..324
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(543..647)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     715..733
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        833..1212
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1213..1416
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1367..1387
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             1417..1592
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    1655..1854
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             2055..2470
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     2400..2422
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     2438..2454
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        2517..2765
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2517..2537
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2688..2707
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2787..2862
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2969..3180
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             3200..3685
                     /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             3695..3751
                     /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             3746..4546
                     /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(3806..3825)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4416..4435
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     LTR             4667..5092
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(5195..5217)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5353..5373)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5358..5687
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5538..5673
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5709..5728
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5809..5826)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5980..6568)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6060..6079)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6739..7057,1..473,474..542))
                     /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"
ORIGIN
        1 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
       61 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
      121 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
      181 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
      241 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca
      301 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
      361 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
      421 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
      481 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
      541 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
      601 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
      661 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
      721 cgtatcacga ggccctttcg tgttcaagaa ttagcttggc cattgcatac gttgtatcca
      781 tatcataata tgtacattta tattggctca tgtccaacat taccgccatg ttgacattga
      841 ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
      901 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
      961 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
     1021 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     1081 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     1141 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     1201 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
     1261 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
     1321 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
     1381 aggcgtgtac ggtgggaggt ctatataagc agagctcaat aaaagagccc acaacccctc
     1441 actcggcgcg ccagtcctcc gatagactgc gtcgcccggg tacccgtatt cccaataaag
     1501 cctcttgctg tttgcatccg aatcgtggtc tcgctgttcc ttgggagggt ctcctctgag
     1561 tgattgacta cccacgacgg gggtctttca tttgggggct cgtccgggat ttggagaccc
     1621 ctgcccaggg accaccgacc caccaccggg aggtaagctg gccagcaact tatctgtgtc
     1681 tgtccgattg tctagtgtct atgtttgatg ttatgcgcct gcgtctgtac tagttagcta
     1741 actagctctg tatctggcgg acccgtggtg gaactgacga gttctgaaca cccggccgca
     1801 accctgggag acgtcccagg gactttgggg gccgtttttg tggcccgacc tgaggaaggg
     1861 agtcgatgtg gaatccgacc ccgtcaggat atgtggttct ggtaggagac gagaacctaa
     1921 aacagttccc gcctccgtct gaatttttgc tttcggtttg gaaccgaagc cgcgcgtctt
     1981 gtctgctgca gcgctgcagc atcgttctgt gttgtctctg tctgactgtg tttctgtatt
     2041 tgtctgaaaa ttagggccag actgttacca ctcccttaag tttgacctta ggtcactgga
     2101 aagatgtcga gcggatcgct cacaaccagt cggtagatgt caagaagaga cgttggttac
     2161 cttctgctct gcagaatggc caacctttaa cgtcggatgg ccgcgagacg gcacctttaa
     2221 ccgagacctc atcacccagg ttaagatcaa ggtcttttca cctggcccgc atggacaccc
     2281 agaccaggtc ccctacatcg tgacctggga agccttggct tttgaccccc ctccctgggt
     2341 caagcccttt gtacacccta agcctccgcc tcctcttcct ccatccgccc cgtctctccc
     2401 ccttgaacct cctcgttcga ccccgcctcg atcctccctt tatccagccc tcactccttc
     2461 tctaggcgcc ggaattgaag atctttaatt aacgatctat cgattctaga tctagagagg
     2521 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
     2581 ttagaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
     2641 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
     2701 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
     2761 acaccgagtc gcgcagcacc gagttggttt tagagctaga aatagcaagt taaaataagg
     2821 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttga atttagatct
     2881 tgagacaagg tgccgggccg ggaattcggc gaattgggcc cgacgtcgca tgctcccggc
     2941 ccctcgagtg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag
     3001 aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac
     3061 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
     3121 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag
     3181 gtgtcgtgac gcggccacca tgaacccagc catcagcgtc gctctcctgc tctcagtctt
     3241 gcaggtgtcc cgagggcaga aggtgaccag cctgacagcc tgcctggtga accaaaacct
     3301 tcgcctggac tgccgccatg agaataacac caaggataac tccatccagc atgagttcag
     3361 cctgacccga gagaagagga agcacgtgct ctcaggcacc ctcgggatac ccgagcacac
     3421 gtaccgctcc cgcgtcaccc tctccaacca gccctatatc aaggtcctta ccctagccaa
     3481 cttcaccacc aaggatgagg gcgactactt ttgtgagctt cgagtctcgg gcgcgaatcc
     3541 catgagctcc aataaaagta tcagtgtgta tagagacaaa ctggtcaagt gtggcggcat
     3601 aagcctgctg gttcagaaca catcctggat gctgctgctg ctgctttccc tctccctcct
     3661 ccaagccctg gacttcattt ctctgggcag cggcgccacc aacttcagcc tgctgaagca
     3721 ggccggcgac gtggaggaga accccggccc catgattgaa caagatggat tgcacgcagg
     3781 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     3841 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     3901 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     3961 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     4021 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     4081 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     4141 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     4201 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     4261 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     4321 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     4381 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     4441 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     4501 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgattag atatcatcag
     4561 tggtccaggc tctagttttg actcaacaat atcaccagct gaagcctata gagtacgagc
     4621 catagataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg aaagacccca
     4681 cctgtaggtt tggcaagcta gcttaagtaa cgccattttg caaggcatgg aaaaatacat
     4741 aactgagaat agagaagttc agatcaaggt caggaacaga tggaacaggg tcgaccctag
     4801 agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg
     4861 aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat
     4921 aaaagagccc acaacccctc actcggggcg ccagtcctcc gattgactga gtcgcccggg
     4981 tacccgtgta tccaataaac cctcttgcag ttgcatccga cttgtggtct cgctgttcct
     5041 tgggagggtc tcctctgagt gattgactac ccgtcagcgg gggtctttca tttgggggct
     5101 cgtccgggat cgggagaccc ctgcccaggg accaccgacc caccaccggg aggtaagctg
     5161 gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     5221 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     5281 cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt
     5341 agatccggct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     5401 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     5461 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     5521 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     5581 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     5641 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag cttactggct
     5701 taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc
     5761 gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga
     5821 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     5881 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     5941 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     6001 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     6061 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     6121 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     6181 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     6241 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     6301 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     6361 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     6421 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     6481 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6541 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     6601 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     6661 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     6721 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     6781 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     6841 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     6901 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     6961 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     7021 agttaatagt ttgcgcaacg ttgttgccat tgctgca
//
