LOCUS       sequence-476301-        8332 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the sequential insertion of two guide RNAs into two U6gRNA 
            cassettes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8332)
  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 8332)
  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-U6gRNA2-Filler
FEATURES             Location/Qualifiers
     source          1..8332
                     /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)"
     misc_RNA        2428..2503
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2532..2780
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        3418..3493
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3516..4015
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3551..3574)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3948..3967
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4040..4741
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     LTR             4849..5363
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5525..5541
                     /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(5549..5555,5556..5573,5574..5579))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5594..5615
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5732..5749)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5903..6491)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5983..6002)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6662..7453,7454..7522))
                     /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     7285..7304
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7523..7627)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7695..7713
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7751..7773)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7873..7892
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8086..8108
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8236..8255
                     /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 cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
     2581 ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata
     2641 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     2701 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
     2761 tgtggaaagg acgaaacacc ggaggcaggt gcaatttgag ttaagggtgt tgctctcaat
     2821 gatttcatta atggttcaat ttttaatttc ttttcttctg gtttaaaatt caagtttaaa
     2881 gtgaaagtgt aatatgcacc catttcttta aataaatctt ctaaatagtc tactaatgtt
     2941 ttattttgtt ttttataaaa tcaagcagcc tctgctatta atatagaagc ttgtattcca
     3001 tctttatctc tagctgagtc atcaattaca tatccataac tttcttcata agcaaaaaca
     3061 aaatttaatc cgttatcttc ttctttagca atttctctac ccattcattt aaatccagtt
     3121 aaagttttta caatattaac tccatatttt tcatgagcga ttctatcacc caaatcactt
     3181 gttacaaaac ttgaatatag agccggattt tttggaatgc tatttaagcg ttttagattt
     3241 gataattttc aatcaattaa aattggtcct gtttgatttc catctaatct tacaaaatga
     3301 ccatcatgtt ttattgccat tccaaatctg tcagcatctg ggtcattcat aataataata
     3361 tctgcatcat gtttaatacc atattcaagc ggtatttttc atgcaggacc tgcctcagtt
     3421 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
     3481 accgagtcgg tgcttttttt ggatccaatt ctaccgggta ggggaggcgc ttttcccaag
     3541 gcagtctgga gcatgcgctt tagcagcccc gctgggcact tggcgctaca caagtggcct
     3601 ctggcctcgc acacattcca catccaccgg taggcgccaa ccggctccgt tctttggtgg
     3661 ccccttcgcg ccaccttcta ctcctcccct agtcaggaag ttcccccccg ccccgcagct
     3721 cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc actagtctcg tgcagatgga
     3781 cagcaccgct gagcaatgga agcgggtagg cctttggggc agcggccaat agcagctttg
     3841 ctccttcgct ttctgggctc agaggctggg aaggggtggg tccgggggcg ggctcagggg
     3901 cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat tctgcacgct
     3961 tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca tctccgggcc tttcggaccg
     4021 gttctagagc gctgccacca tgagcgagct gattaaggag aacatgcaca tgaagctgta
     4081 catggagggc accgtggaca accatcactt caagtgcaca tccgagggcg aaggcaagcc
     4141 ctacgagggc acccagacca tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc
     4201 cttcgacatc ctggctacta gcttcctcta cggcagcaag accttcatca accacaccca
     4261 gggcatcccc gacttcttca agcagtcctt ccctgagggc ttcacatggg agagagtcac
     4321 cacatacgag gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg
     4381 cctcatctac aacgtcaaga tcagaggggt gaacttcaca tccaacggcc ctgtgatgca
     4441 gaagaaaaca ctcggctggg aggccttcac cgagacgctg taccccgctg acggcggcct
     4501 ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg agccatctga tcgcaaacat
     4561 caagaccaca tatagatcca agaaacccgc taagaacctc aagatgcctg gcgtctacta
     4621 tgtggactac agactggaaa gaatcaagga ggccaacaac gagacctacg tcgagcagca
     4681 cgaggtggca gtggccagat actgcgacct ccctagcaaa ctggggcaca agcttaattg
     4741 agcggccgct gaattaatta atatcaagct tatcgatacc gtcgactcct gcagccaagc
     4801 ttatcgataa aataaaagat tttatttagt ctccagaaaa aggggggaat gaaagacccc
     4861 acctgtaggt ttggcaagct agcttaagta acgccatttt gcaaggcatg gaaaatacat
     4921 aactgagaat agagaagttc agatcaaggt taggaacaga gagacagcag aatatgggcc
     4981 aaacaggata tctgtggtaa gcagttcctg ccccggctca gggccaagaa cagatggtcc
     5041 ccagatgcgg tcccgccctc agcagtttct agagaaccat cagatgtttc cagggtgccc
     5101 caaggacctg aaatgaccct gtgccttatt tgaactaacc aatcagttcg cttctcgctt
     5161 ctgttcgcgc gcttctgctc cccgagctca ataaaagagc ccacaacccc tcactcggcg
     5221 cgccagtcct ccgatagact gcgtcgcccg ggtacccgtg tatccaataa accctcttgc
     5281 agttgcatcc gacttgtggt ctcgctgttc cttgggaggg tctcctctga gtgattgact
     5341 acccgtcagc gggggtcttt catgggtaac agtttcttga agttggagaa caacattctg
     5401 agggtaggag tcgaatatta agtaatcctg actcaattag ccactgtttt gaatccacat
     5461 actccaatac tcctgaaata gttcattatg gacagcgcag aaagagctgg ggagaattgt
     5521 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     5581 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     5641 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     5701 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     5761 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     5821 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     5881 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5941 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     6001 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     6061 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     6121 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     6181 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     6241 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     6301 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     6361 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     6421 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     6481 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     6541 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     6601 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     6661 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     6721 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     6781 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     6841 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6901 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6961 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     7021 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     7081 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     7141 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     7201 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     7261 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     7321 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     7381 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     7441 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     7501 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     7561 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     7621 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     7681 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg
     7741 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     7801 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct
     7861 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa
     7921 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc
     7981 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
     8041 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
     8101 gtaaaacgac ggcgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     8161 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     8221 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
     8281 ggtgatgccg gccacgatgc gtccggcgta gaggcgatta gtccaatttg tt
//
