LOCUS       sequence-476302-        7354 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-v1..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7354)
  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 7354)
  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-v2
FEATURES             Location/Qualifiers
     source          1..7354
                     /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        1558..1806
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1558..1578
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1729..1748
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2440..2515
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2538..3037
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2573..2596)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2970..2989
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3062..3763
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     LTR             3871..4385
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4547..4563
                     /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(4571..4577,4578..4595,4596..4601))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4616..4637
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4754..4771)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4925..5513)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5005..5024)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5684..6475,6476..6544))
                     /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     6307..6326
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6545..6649)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6717..6735
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6773..6795)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6895..6914
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7108..7130
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7258..7277
                     /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 tcgaggttaa cgaattcgag
     1561 ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata
     1621 attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag
     1681 taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg
     1741 cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga
     1801 aacaccgggt cttcgcaatt tgagttaagg gtgttgctct caatgatttc attaatggtt
     1861 caatttttaa tttcttttct tctggtttaa aattcaagtt taaagtgaaa gtgtaatatg
     1921 cacccatttc tttaaataaa tcttctaaat agtctactaa tgttttattt tgttttttat
     1981 aaaatcaagc agcctctgct attaatatag aagcttgtat tccatcttta tctctagctg
     2041 agtcatcaat tacatatcca taactttctt cataagcaaa aacaaaattt aatccgttat
     2101 cttcttcttt agcaatttct ctacccattc atttaaatcc agttaaagtt tttacaatat
     2161 taactccata tttttcatga gcgattctat cacccaaatc acttgttaca aaacttgaat
     2221 atagagccgg attttttgga atgctattta agcgttttag atttgataat tttcaatcaa
     2281 ttaaaattgg tcctgtttga tttccatcta atcttacaaa atgaccatca tgttttattg
     2341 ccattccaaa tctgtcagca tctgggtcat tcataataat aatatctgca tcatgtttaa
     2401 taccatattc aagcggtatt tttcatgcag ggaagacctg ttttagagct agaaatagca
     2461 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     2521 ttggatccaa ttctaccggg taggggaggc gcttttccca aggcagtctg gagcatgcgc
     2581 tttagcagcc ccgctgggca cttggcgcta cacaagtggc ctctggcctc gcacacattc
     2641 cacatccacc ggtaggcgcc aaccggctcc gttctttggt ggccccttcg cgccaccttc
     2701 tactcctccc ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg
     2761 acaaatggaa gtagcacgtc tcactagtct cgtgcagatg gacagcaccg ctgagcaatg
     2821 gaagcgggta ggcctttggg gcagcggcca atagcagctt tgctccttcg ctttctgggc
     2881 tcagaggctg ggaaggggtg ggtccggggg cgggctcagg ggcgggctca ggggcggggc
     2941 gggcgcccga aggtcctccg gaggcccggc attctgcacg cttcaaaagc gcacgtctgc
     3001 cgcgctgttc tcctcttcct catctccggg cctttcggac cggttctaga gcgctgccac
     3061 catgagcgag ctgattaagg agaacatgca catgaagctg tacatggagg gcaccgtgga
     3121 caaccatcac ttcaagtgca catccgaggg cgaaggcaag ccctacgagg gcacccagac
     3181 catgagaatc aaggtggtcg agggcggccc tctccccttc gccttcgaca tcctggctac
     3241 tagcttcctc tacggcagca agaccttcat caaccacacc cagggcatcc ccgacttctt
     3301 caagcagtcc ttccctgagg gcttcacatg ggagagagtc accacatacg aggacggggg
     3361 cgtgctgacc gctacccagg acaccagcct ccaggacggc tgcctcatct acaacgtcaa
     3421 gatcagaggg gtgaacttca catccaacgg ccctgtgatg cagaagaaaa cactcggctg
     3481 ggaggccttc accgagacgc tgtaccccgc tgacggcggc ctggaaggca gaaacgacat
     3541 ggccctgaag ctcgtgggcg ggagccatct gatcgcaaac atcaagacca catatagatc
     3601 caagaaaccc gctaagaacc tcaagatgcc tggcgtctac tatgtggact acagactgga
     3661 aagaatcaag gaggccaaca acgagaccta cgtcgagcag cacgaggtgg cagtggccag
     3721 atactgcgac ctccctagca aactggggca caagcttaat tgagcggccg ctgaattaat
     3781 taatatcaag cttatcgata ccgtcgactc ctgcagccaa gcttatcgat aaaataaaag
     3841 attttattta gtctccagaa aaagggggga atgaaagacc ccacctgtag gtttggcaag
     3901 ctagcttaag taacgccatt ttgcaaggca tggaaaatac ataactgaga atagagaagt
     3961 tcagatcaag gttaggaaca gagagacagc agaatatggg ccaaacagga tatctgtggt
     4021 aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc ggtcccgccc
     4081 tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc tgaaatgacc
     4141 ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc
     4201 tccccgagct caataaaaga gcccacaacc cctcactcgg cgcgccagtc ctccgataga
     4261 ctgcgtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat ccgacttgtg
     4321 gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca gcgggggtct
     4381 ttcatgggta acagtttctt gaagttggag aacaacattc tgagggtagg agtcgaatat
     4441 taagtaatcc tgactcaatt agccactgtt ttgaatccac atactccaat actcctgaaa
     4501 tagttcatta tggacagcgc agaaagagct ggggagaatt gtgaaattgt tatccgctca
     4561 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4621 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4681 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4741 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4801 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4861 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4921 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4981 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     5041 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5101 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5161 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5221 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5281 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5341 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5401 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5461 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5521 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5581 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5641 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5701 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5761 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5821 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5881 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5941 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     6001 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6061 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6121 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6181 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6241 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6301 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6361 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6421 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6481 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6541 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6601 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6661 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
     6721 ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac
     6781 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     6841 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat
     6901 cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa
     6961 ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc
     7021 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
     7081 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggcgcaag
     7141 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat
     7201 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
     7261 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
     7321 gcgtccggcg tagaggcgat tagtccaatt tgtt
//
