LOCUS       sequence-476304-        7366 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For inserting a single guide RNA or later cloning two U6-gRNA 
            cassettes with MIGR1-U6gRNA1-Filler-v2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7366)
  AUTHORS   Zhong X, Wu H, Wang G, Sun Z
  TITLE     Protocol for CRISPR-mediated deletion of cis-regulatory element in 
            murine Th17 cells for in vivo assessment of effector function.
  JOURNAL   STAR Protoc. 2025 Jun 20;6(2):103831. doi: 
            10.1016/j.xpro.2025.103831. Epub 2025 May 20.
  PUBMED    40397576
REFERENCE   2  (bases 1 to 7366)
  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: "STAR 
            Protoc."; date: "2025-06-20"; volume: "6(2):103831. doi"; pages: " 
            10.1016/j.xpro.2025.103831. Epub 2025 May 20"
COMMENT     Sequence Label: MIGR1-U6gRNA1-Filler-v1
FEATURES             Location/Qualifiers
     source          1..7366
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             123..639
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    703..1044
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             1111..1527
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1457..1479
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1495..1511
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        1546..1794
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1546..1566
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1717..1736
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2428..2503
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2550..3049
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2585..2608)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2982..3001
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3074..3775
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     LTR             3883..4397
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4559..4575
                     /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)."
     promoter        complement(join(4583..4589,4590..4607,4608..4613))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4628..4649
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4766..4783)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4937..5525)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5017..5036)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5696..6487,6488..6556))
                     /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     6319..6338
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6557..6661)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6729..6747
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6785..6807)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6907..6926
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7120..7142
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7270..7289
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 aaagacagga tatcagtggt ccaggctcta gttttgactc aacaatatca ccagctgaag
       61 cctatagagt acgagccata gataaaataa aagattttat ttagtctcca gaaaaagggg
      121 ggaatgaaag accccacctg taggtttggc aagctagctt aagtaacgcc attttgcaag
      181 gcatggaaaa tacataactg agaatagaga agttcagatc aaggttagga acagagagac
      241 agcagaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc
      301 aagaacagat ggtccccaga tgcggtcccg ccctcagcag tttctagaga accatcagat
      361 gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac taaccaatca
      421 gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa agagcccaca
      481 acccctcact cggcgcgcca gtcctccgat agactgcgtc gcccgggtac ccgtattccc
      541 aataaagcct cttgctgttt gcatccgaat cgtggactcg ctgatccttg ggagggtctc
      601 ctcagattga ttgactgccc acctcggggg tctttcattt ggaggttcca ccgagatttg
      661 gagacccctg cccagggacc accgaccccc ccgccgggag gtaagctggc cagcggtcgt
      721 ttcgtgtctg tctctgtctt tgtgcgtgtt tgtgccggca tctaatgttt gcgcctgcgt
      781 ctgtactagt tagctaacta gctctgtatc tggcggaccc gtggtggaac tgacgagttc
      841 tgaacacccg gccgcaaccc tgggagacgt cccagggact ttgggggccg tttttgtggc
      901 ccgacctgag gaagggagtc gatgtggaat ccgaccccgt caggatatgt ggttctggta
      961 ggagacgaga acctaaaaca gttcccgcct ccgtctgaat ttttgctttc ggtttggaac
     1021 cgaagccgcg cgtcttgtct gctgcagcgc tgcagcatcg ttctgtgttg tctctgtctg
     1081 actgtgtttc tgtatttgtc tgaaaattag ggccagactg ttaccactcc cttaagtttg
     1141 accttaggtc actggaaaga tgtcgagcgg atcgctcaca accagtcggt agatgtcaag
     1201 aagagacgtt gggttacctt ctgctctgca gaatggccaa cctttaacgt cggatggccg
     1261 cgagacggca cctttaaccg agacctcatc acccaggtta agatcaaggt cttttcacct
     1321 ggcccgcatg gacacccaga ccaggtcccc tacatcgtga cctgggaagc cttggctttt
     1381 gacccccctc cctgggtcaa gccctttgta caccctaagc ctccgcctcc tcttcctcca
     1441 tccgccccgt ctctccccct tgaacctcct cgttcgaccc cgcctcgatc ctccctttat
     1501 ccagccctca ctccttctct aggcgccgga attagatctc tcgaggaggg cctatttccc
     1561 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tagaattaat
     1621 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     1681 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     1741 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgggtct
     1801 tcgcaatttg agttaagggt gttgctctca atgatttcat taatggttca atttttaatt
     1861 tcttttcttc tggtttaaaa ttcaagttta aagtgaaagt gtaatatgca cccatttctt
     1921 taaataaatc ttctaaatag tctactaatg ttttattttg ttttttataa aatcaagcag
     1981 cctctgctat taatatagaa gcttgtattc catctttatc tctagctgag tcatcaatta
     2041 catatccata actttcttca taagcaaaaa caaaatttaa tccgttatct tcttctttag
     2101 caatttctct acccattcat ttaaatccag ttaaagtttt tacaatatta actccatatt
     2161 tttcatgagc gattctatca cccaaatcac ttgttacaaa acttgaatat agagccggat
     2221 tttttggaat gctatttaag cgttttagat ttgataattt tcaatcaatt aaaattggtc
     2281 ctgtttgatt tccatctaat cttacaaaat gaccatcatg ttttattgcc attccaaatc
     2341 tgtcagcatc tgggtcattc ataataataa tatctgcatc atgtttaata ccatattcaa
     2401 gcggtatttt tcatgcaggg aagacctgtt ttagagctag aaatagcaag ttaaaataag
     2461 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gcacagcgcg
     2521 tatgtgaatt cggtggatcc aattctaccg ggtaggggag gcgcttttcc caaggcagtc
     2581 tggagcatgc gctttagcag ccccgctggg cacttggcgc tacacaagtg gcctctggcc
     2641 tcgcacacat tccacatcca ccggtaggcg ccaaccggct ccgttctttg gtggcccctt
     2701 cgcgccacct tctactcctc ccctagtcag gaagttcccc cccgccccgc agctcgcgtc
     2761 gtgcaggacg tgacaaatgg aagtagcacg tctcactagt ctcgtgcaga tggacagcac
     2821 cgctgagcaa tggaagcggg taggcctttg gggcagcggc caatagcagc tttgctcctt
     2881 cgctttctgg gctcagaggc tgggaagggg tgggtccggg ggcgggctca ggggcgggct
     2941 caggggcggg gcgggcgccc gaaggtcctc cggaggcccg gcattctgca cgcttcaaaa
     3001 gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg ggcctttcgg accggttcta
     3061 gagcgctgcc accatgagcg agctgattaa ggagaacatg cacatgaagc tgtacatgga
     3121 gggcaccgtg gacaaccatc acttcaagtg cacatccgag ggcgaaggca agccctacga
     3181 gggcacccag accatgagaa tcaaggtggt cgagggcggc cctctcccct tcgccttcga
     3241 catcctggct actagcttcc tctacggcag caagaccttc atcaaccaca cccagggcat
     3301 ccccgacttc ttcaagcagt ccttccctga gggcttcaca tgggagagag tcaccacata
     3361 cgaggacggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat
     3421 ctacaacgtc aagatcagag gggtgaactt cacatccaac ggccctgtga tgcagaagaa
     3481 aacactcggc tgggaggcct tcaccgagac gctgtacccc gctgacggcg gcctggaagg
     3541 cagaaacgac atggccctga agctcgtggg cgggagccat ctgatcgcaa acatcaagac
     3601 cacatataga tccaagaaac ccgctaagaa cctcaagatg cctggcgtct actatgtgga
     3661 ctacagactg gaaagaatca aggaggccaa caacgagacc tacgtcgagc agcacgaggt
     3721 ggcagtggcc agatactgcg acctccctag caaactgggg cacaagctta attgagcggc
     3781 cgctgaatta attaatatca agcttatcga taccgtcgac tcctgcagcc aagcttatcg
     3841 ataaaataaa agattttatt tagtctccag aaaaaggggg gaatgaaaga ccccacctgt
     3901 aggtttggca agctagctta agtaacgcca ttttgcaagg catggaaaat acataactga
     3961 gaatagagaa gttcagatca aggttaggaa cagagagaca gcagaatatg ggccaaacag
     4021 gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg gtccccagat
     4081 gcggtcccgc cctcagcagt ttctagagaa ccatcagatg tttccagggt gccccaagga
     4141 cctgaaatga ccctgtgcct tatttgaact aaccaatcag ttcgcttctc gcttctgttc
     4201 gcgcgcttct gctccccgag ctcaataaaa gagcccacaa cccctcactc ggcgcgccag
     4261 tcctccgata gactgcgtcg cccgggtacc cgtgtatcca ataaaccctc ttgcagttgc
     4321 atccgacttg tggtctcgct gttccttggg agggtctcct ctgagtgatt gactacccgt
     4381 cagcgggggt ctttcatggg taacagtttc ttgaagttgg agaacaacat tctgagggta
     4441 ggagtcgaat attaagtaat cctgactcaa ttagccactg ttttgaatcc acatactcca
     4501 atactcctga aatagttcat tatggacagc gcagaaagag ctggggagaa ttgtgaaatt
     4561 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     4621 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     4681 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4741 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4801 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4861 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4921 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4981 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5041 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5101 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5161 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5221 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5281 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5341 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5401 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5461 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5521 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5581 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5641 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5701 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5761 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5821 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5881 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5941 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6001 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6061 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6121 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6181 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6241 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6301 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6361 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6421 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6481 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6541 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6601 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6661 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     6721 cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg
     6781 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     6841 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
     6901 actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc
     6961 acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg ctgcgcaact
     7021 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     7081 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
     7141 cgacggcgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     7201 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     7261 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     7321 gccggccacg atgcgtccgg cgtagaggcg attagtccaa tttgtt
//
