LOCUS       sequence-364054-        7056 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 7056)
  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 7056)
  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-gRNA2
FEATURES             Location/Qualifiers
     source          1..7056
                     /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        2786..2861
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2968..3179
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             3199..3684
                     /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             3694..3750
                     /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             3745..4545
                     /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(3805..3824)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4415..4434
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     LTR             4666..5091
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(5194..5216)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5352..5372)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5357..5686
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5537..5672
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5708..5727
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5808..5825)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5979..6567)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6059..6078)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6738..7056,1..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 acaccggtac atgaggcggc agtcggtttt agagctagaa atagcaagtt aaaataaggc
     2821 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgaa tttagatctt
     2881 gagacaaggt gccgggccgg gaattcggcg aattgggccc gacgtcgcat gctcccggcc
     2941 cctcgagtgg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga
     3001 agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc ggggtaaact
     3061 gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata
     3121 taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg
     3181 tgtcgtgacg cggccaccat gaacccagcc atcagcgtcg ctctcctgct ctcagtcttg
     3241 caggtgtccc gagggcagaa ggtgaccagc ctgacagcct gcctggtgaa ccaaaacctt
     3301 cgcctggact gccgccatga gaataacacc aaggataact ccatccagca tgagttcagc
     3361 ctgacccgag agaagaggaa gcacgtgctc tcaggcaccc tcgggatacc cgagcacacg
     3421 taccgctccc gcgtcaccct ctccaaccag ccctatatca aggtccttac cctagccaac
     3481 ttcaccacca aggatgaggg cgactacttt tgtgagcttc gagtctcggg cgcgaatccc
     3541 atgagctcca ataaaagtat cagtgtgtat agagacaaac tggtcaagtg tggcggcata
     3601 agcctgctgg ttcagaacac atcctggatg ctgctgctgc tgctttccct ctccctcctc
     3661 caagccctgg acttcatttc tctgggcagc ggcgccacca acttcagcct gctgaagcag
     3721 gccggcgacg tggaggagaa ccccggcccc atgattgaac aagatggatt gcacgcaggt
     3781 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     3841 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     3901 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     3961 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     4021 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     4081 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     4141 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     4201 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     4261 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     4321 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     4381 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     4441 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     4501 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgattaga tatcatcagt
     4561 ggtccaggct ctagttttga ctcaacaata tcaccagctg aagcctatag agtacgagcc
     4621 atagataaaa taaaagattt tatttagtct ccagaaaaag gggggaatga aagaccccac
     4681 ctgtaggttt ggcaagctag cttaagtaac gccattttgc aaggcatgga aaaatacata
     4741 actgagaata gagaagttca gatcaaggtc aggaacagat ggaacagggt cgaccctaga
     4801 gaaccatcag atgtttccag ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga
     4861 actaaccaat cagttcgctt ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata
     4921 aaagagccca caacccctca ctcggggcgc cagtcctccg attgactgag tcgcccgggt
     4981 acccgtgtat ccaataaacc ctcttgcagt tgcatccgac ttgtggtctc gctgttcctt
     5041 gggagggtct cctctgagtg attgactacc cgtcagcggg ggtctttcat ttgggggctc
     5101 gtccgggatc gggagacccc tgcccaggga ccaccgaccc accaccggga ggtaagctgg
     5161 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     5221 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     5281 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta
     5341 gatccggctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     5401 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     5461 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     5521 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     5581 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     5641 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc ttactggctt
     5701 aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg
     5761 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
     5821 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     5881 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5941 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     6001 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     6061 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     6121 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     6181 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     6241 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     6301 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     6361 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     6421 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     6481 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     6541 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6601 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     6661 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     6721 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     6781 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6841 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     6901 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     6961 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     7021 gttaatagtt tgcgcaacgt tgttgccatt gctgca
//
