LOCUS       sequence-375686-        6517 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral delivery of guide RNA against mouse Runx1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6517)
  AUTHORS   Shin B, Hosokawa H, Romero-Wolf M, Zhou W, Masuhara K, Tobin VR, 
            Levanon D, Groner Y, Rothenberg EV
  TITLE     Runx1 and Runx3 drive progenitor to T-lineage transcriptome 
            conversion in mouse T cell commitment via dynamic genomic site 
            switching.
  JOURNAL   Proc Natl Acad Sci U S A. 2021 Jan 26;118(4). pii: 2019655118. doi: 
            10.1073/pnas.2019655118.
  PUBMED    33479171
REFERENCE   2  (bases 1 to 6517)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2021-01-26"; volume: "118(4). pii: 
            2019655118. doi"; pages: " 10.1073/pnas.2019655118"
COMMENT     Sequence Label: E42_gRunx1_gRNA2_dTet_NGFR
FEATURES             Location/Qualifiers
     source          1..6517
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     181..201
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     primer_bind     337..359
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     LTR             462..887
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     regulatory      1902..1911
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1928..2139)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     promoter        complement(2279..2527)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2337..2356)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2507..2527)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(2543..2959)
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     complement(2559..2575)
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     complement(2591..2613)
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     misc_feature    3160..3359
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     LTR             3422..3597
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     promoter        complement(3598..3801)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3627..3647)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3802..4181
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(4281..4299)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4367..4471
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4472..4540,4541..5332)
                     /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"
     primer_bind     complement(4690..4709)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5503..6091
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5992..6011
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6245..6262
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(6343..6362)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        complement(join(6384..6517,1..196))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(6398..6517,1..16)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 gagttagggg cgggatgggc ggagttaggg gcgggactat ggttgctgac taattgagat
       61 gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac ctggttgctg
      121 actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac
      181 accctaactg acacacattc cacagccgga tctacactcc gctatcgcta cgtgactggg
      241 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
      301 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
      361 tttcaccgtc atcaccgaaa cgcgcgaggc agccagctta cctcccggtg gtgggtcggt
      421 ggtccctggg caggggtctc ccgatcccgg acgagccccc aaatgaaaga cccccgctga
      481 cgggtagtca atcactcaga ggagaccctc ccaaggaaca gcgagaccac aagtcggatg
      541 caactgcaag agggtttatt ggatacacgg gtacccgggc gactcagtca atcggaggac
      601 tggcgccccg agtgaggggt tgtgggctct tttattgagc tcggggagca gaagcgcgcg
      661 aacagaagcg agaagcgaac tgattggtta gttcaaataa ggcacagggt catttcaggt
      721 ccttggggca ccctggaaac atctgatggt tctctagggt cgaccctgtt ccatctgttc
      781 ctgaccttga tctgaacttc tctattctca gttatgtatt tttccatgcc ttgcaaaatg
      841 gcgttactta agctagcttg ccaaacctac aggtggggtc tttcattccc ccctttttct
      901 ggagactaaa taaaatcttt tattttatct atggctcgta ctctataggc ttcagctggt
      961 gatattgttg agtcaaaact agagcctgga ccactgatat cctgtcttta acaaattgga
     1021 ctaatcgatt tatcgataag cttggctgca ggtcgactct agaccgcggt gacctcgagg
     1081 atatcgatga ctgttccacc tcttgaaggc tatgtaggcc acaaggccca caaccacagc
     1141 agccaggatg gagcaataga cagggatgag gttgtcggtg gtgcctcggg tcaccacggg
     1201 ctgggagctg cccatcactg tggtcaccac acctgccacc gtgctggcta tgaggtcttg
     1261 ttctggaggt gcctcaggct cctgggtgct gggggctgtg ctgtccgagc cctctggggg
     1321 tgtggaccgt gtaatccaac ggccagggat ctcctcgcac tcggcgtcgg cccagcgtgt
     1381 gcactcgcgg agctggcgct cggtgtcctc gcacacggtg cagggcaggc acgggtccac
     1441 gtggttggcc tcgtcggaat acgtgccgtc ggggcactcc tcgcacacgg tgttctgctt
     1501 gtcctggcag gagaacacga ggcccgagcc cgcctcgcac acgcggcacg cctcgcagcg
     1561 cccagtcgtc tcatcctggt agtagccgta ggcgcagcgg cacacggcgt cgtcggcctc
     1621 cacgcacggc gccgacatgc tctggagccc cacgcactcg gtgcacggct tgcacggctc
     1681 ggtcgcgctc accacgtcgg agaacgtcac gctgtccagg cagggctcac acacggtctg
     1741 gttggctcca caaggctggg ccacaccctc gcccaggttg caggctttgc agcactcacc
     1801 gctgtgtgtg tacaggcctg tggggcatgc ctccttggca cctccaaggg acacccccag
     1861 aagcagcaac agcagcaggc gcggcccgtc catggttgtg gccatggtgg cgaccgcgtc
     1921 acgacacctg tgttctggcg gcaaacccgt tgcgaaaaag aacgttcacg gcgactactg
     1981 cacttatata cggttctccc ccaccctcgg gaaaaaggcg gagccagtac acgacatcac
     2041 tttcccagtt taccccgcgc caccttctct aggcaccggt tcaattgccg acccctcccc
     2101 ccaacttctc ggggactgtg ggcgatgtgc gctctgccca ctgacgggca ccggagccac
     2161 tcgagaaaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat
     2221 ttaaacttgc tatgctgttt ccagcatagc tcttaaaccg ctcatcttgc cggggctcgg
     2281 tgtttcgtcc tttccacaag atatataaag ccaagaaatc gaaatacttt caagttacgg
     2341 taagcatatg atagtccatt ttaaaacata attttaaaac tgcaaactac ccaagaaatt
     2401 attactttct acgtcacgta ttttgtacta atatctttgt gtttacagtc aaattaattc
     2461 caattatctc tctaacagcc ttgtatcgta tatgcaaata tgaaggaatc atgggaaata
     2521 ggccctcgga tccttcaatt ccggcgccta gagaaggagt gagggctgga taaagggagg
     2581 atcgaggcgg ggtcgaacga ggaggttcaa gggggagaga cggggcggat ggaggaagag
     2641 gaggcggagg cttagggtgt acaaagggct tgacccaggg aggggggtca aaagccaagg
     2701 cttcccaggt cacgatgtag gggacctggt ctgggtgtcc atgcgggcca ggtgaaaaga
     2761 ccttgatctt aacctgggtg atgaggtctc ggttaaaggt gccgtctcgc ggccatccga
     2821 cgttaaaggt tggccattct gcagagcaga aggtaaccca acgtctcttc ttgacatcta
     2881 ccgactggtt gtgagcgatc cgctcgacat ctttccagtg acctaaggtc aaacttaagg
     2941 gagtggtaac agtctggccc taattttcag acaaatacag aaacacagtc agacagagac
     3001 aacacagaac gatgctgcag cgctgcagca gacaagacgc gcggcttcgg ttccaaaccg
     3061 aaagcaaaaa ttcagacgga ggcgggaact gttttaggtt ctcgtctcct accagaacca
     3121 catatcctga cggggtcgga ttccacatcg actcccttcc tcaggtcggg ccacaaaaac
     3181 ggcccccaaa gtccctggga cgtctcccag ggttgcggcc gggtgttcag aactcgtcag
     3241 ttccaccacg ggtccgccag atacagagct agttagctaa ctagtacaga cgcaggcgca
     3301 taacatcaaa catagacact agacaatcgg acagacacag ataagttgct ggccagctta
     3361 cctcccggtg gtgggtcggt ggtccctggg caggggtctc caaatcccgg acgagccccc
     3421 aaatgaaaga cccccgtcgt gggtagtcaa tcactcagag gagaccctcc caaggaacag
     3481 cgagaccacg attcggatgc aaacagcaag aggctttatt gggaatacgg gtacccgggc
     3541 gacgcagtct atcggaagac tggcgcgccg agtgaggggt tgtgggctct tttattgagc
     3601 tctgcttata tagacctccc accgtacacg cctaccgccc atttgcgtca atggggcgga
     3661 gttgttacga cattttggaa agtcccgttg attttggtgc caaaacaaac tcccattgac
     3721 gtcaatgggg tggagacttg gaaatccccg tgagtcaaac cgctatccac gcccattgat
     3781 gtactgccaa aaccgcatca ccatggtaat agcgatgact aatacgtaga tgtactgcca
     3841 agtaggaaag tcccataagg tcatgtactg ggcataatgc caggcgggcc atttaccgtc
     3901 attgacgtca atagggggcg tacttggcat atgatacact tgatgtactg ccaagtgggc
     3961 agtttaccgt aaatactcca cccattgacg tcaatggaaa gtccctattg gcgttactat
     4021 gggaacatac gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc agccaggcgg
     4081 gccatttacc gtaagttatg taacgcggaa ctccatatat gggctatgaa ctaatgaccc
     4141 cgtaattgat tactattaat aactagtcaa taatcaatgt caacatggcg gtaatgttgg
     4201 acatgagcca atataaatgt acatattatg atatggatac aacgtatgca atggccaagc
     4261 taattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     4321 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4381 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     4441 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     4501 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     4561 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     4621 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     4681 taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg
     4741 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     4801 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     4861 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     4921 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     4981 cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa
     5041 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     5101 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     5161 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     5221 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     5281 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     5341 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     5401 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     5461 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     5521 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     5581 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     5641 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     5701 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     5761 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     5821 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     5881 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     5941 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     6001 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     6061 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     6121 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     6181 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     6241 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     6301 gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc
     6361 cgcatagtta agccagtaag ctttttgcaa aagcctaggc ctccaaaaaa gcctcctcac
     6421 tacttctgga atagctcaga ggccgaggcg gcctcggcct ctgcataaat aaaaaaaatt
     6481 agtcagccat ggggcggaga atgggcggaa ctgggcg
//
