LOCUS       sequence-375717-        6361 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral delivery of guide RNA against mouse Runx3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6361)
  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 6361)
  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_gRunx3_gRNA3_dTet_mTurquoise2
FEATURES             Location/Qualifiers
     source          1..6361
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(20..39)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        complement(61..390)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      75..210
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     375..395
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     primer_bind     531..553
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     LTR             656..1081
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     CDS             complement(join(1224..1937,1938..1940,1941..1943))
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1269..1290)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1618..1637
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1877..1898
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1940..1949
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1966..2177)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     promoter        complement(2317..2565)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2375..2394)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2545..2565)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(2581..2997)
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     complement(2597..2613)
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     complement(2629..2651)
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     misc_feature    3198..3397
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     LTR             3460..3635
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     promoter        complement(3636..3839)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3665..3685)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3840..4219
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(4319..4337)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4405..4509
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4510..4578,4579..5370)
                     /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(4728..4747)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5541..6129
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6030..6049
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6283..6300
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtaagctt
       61 tttgcaaaag cctaggcctc caaaaaagcc tcctcactac ttctggaata gctcagaggc
      121 cgaggcggcc tcggcctctg cataaataaa aaaaattagt cagccatggg gcggagaatg
      181 ggcggaactg ggcggagtta ggggcgggat gggcggagtt aggggcggga ctatggttgc
      241 tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc
      301 acacctggtt gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc
      361 tggggacttt ccacacccta actgacacac attccacagc cggatctaca ctccgctatc
      421 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
      481 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
      541 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagccag cttacctccc
      601 ggtggtgggt cggtggtccc tgggcagggg tctcccgatc ccggacgagc ccccaaatga
      661 aagacccccg ctgacgggta gtcaatcact cagaggagac cctcccaagg aacagcgaga
      721 ccacaagtcg gatgcaactg caagagggtt tattggatac acgggtaccc gggcgactca
      781 gtcaatcgga ggactggcgc cccgagtgag gggttgtggg ctcttttatt gagctcgggg
      841 agcagaagcg cgcgaacaga agcgagaagc gaactgattg gttagttcaa ataaggcaca
      901 gggtcatttc aggtccttgg ggcaccctgg aaacatctga tggttctcta gggtcgaccc
      961 tgttccatct gttcctgacc ttgatctgaa cttctctatt ctcagttatg tatttttcca
     1021 tgccttgcaa aatggcgtta cttaagctag cttgccaaac ctacaggtgg ggtctttcat
     1081 tccccccttt ttctggagac taaataaaat cttttatttt atctatggct cgtactctat
     1141 aggcttcagc tggtgatatt gttgagtcaa aactagagcc tggaccactg atatcctgtc
     1201 tttaacaaat tggactaatc gatttacttg tacagctcgt ccatgccgag agtgatcccg
     1261 gcggcggtca cgaactccag caggaccatg tgatcgcgct tctcgttggg gtctttgctc
     1321 agcttggact gggtgctcag gtagtggttg tcgggcagca gcacggggcc gtcgccgatg
     1381 ggggtgttct gctggtagtg gtcggcgagc tgcacgccgc cgtcctcgat gttgtggcgg
     1441 atcttgaagt tggccttgat gccgttcttc tgcttgtcgg cggtgatata gacgttgtcg
     1501 ctaaagtagt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg
     1561 cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc
     1621 ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg
     1681 gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcgggcgaa gcactgcacg
     1741 ccccaggaca gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag
     1801 atgaacttca gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg
     1861 aacttgtggc cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac
     1921 agctcctcgc ccttgctcac catggtggcg accgcgtcac gacacctgtg ttctggcggc
     1981 aaacccgttg cgaaaaagaa cgttcacggc gactactgca cttatatacg gttctccccc
     2041 accctcggga aaaaggcgga gccagtacac gacatcactt tcccagttta ccccgcgcca
     2101 ccttctctag gcaccggttc aattgccgac ccctcccccc aacttctcgg ggactgtggg
     2161 cgatgtgcgc tctgcccact gacgggcacc ggagccactc gagaaaaaaa gcaccgactc
     2221 ggtgccactt tttcaagttg ataacggact agccttattt aaacttgcta tgctgtttcc
     2281 agcatagctc ttaaacccga gctgcgcaac gcttccggtg tttcgtcctt tccacaagat
     2341 atataaagcc aagaaatcga aatactttca agttacggta agcatatgat agtccatttt
     2401 aaaacataat tttaaaactg caaactaccc aagaaattat tactttctac gtcacgtatt
     2461 ttgtactaat atctttgtgt ttacagtcaa attaattcca attatctctc taacagcctt
     2521 gtatcgtata tgcaaatatg aaggaatcat gggaaatagg ccctcggatc cttcaattcc
     2581 ggcgcctaga gaaggagtga gggctggata aagggaggat cgaggcgggg tcgaacgagg
     2641 aggttcaagg gggagagacg gggcggatgg aggaagagga ggcggaggct tagggtgtac
     2701 aaagggcttg acccagggag gggggtcaaa agccaaggct tcccaggtca cgatgtaggg
     2761 gacctggtct gggtgtccat gcgggccagg tgaaaagacc ttgatcttaa cctgggtgat
     2821 gaggtctcgg ttaaaggtgc cgtctcgcgg ccatccgacg ttaaaggttg gccattctgc
     2881 agagcagaag gtaacccaac gtctcttctt gacatctacc gactggttgt gagcgatccg
     2941 ctcgacatct ttccagtgac ctaaggtcaa acttaaggga gtggtaacag tctggcccta
     3001 attttcagac aaatacagaa acacagtcag acagagacaa cacagaacga tgctgcagcg
     3061 ctgcagcaga caagacgcgc ggcttcggtt ccaaaccgaa agcaaaaatt cagacggagg
     3121 cgggaactgt tttaggttct cgtctcctac cagaaccaca tatcctgacg gggtcggatt
     3181 ccacatcgac tcccttcctc aggtcgggcc acaaaaacgg cccccaaagt ccctgggacg
     3241 tctcccaggg ttgcggccgg gtgttcagaa ctcgtcagtt ccaccacggg tccgccagat
     3301 acagagctag ttagctaact agtacagacg caggcgcata acatcaaaca tagacactag
     3361 acaatcggac agacacagat aagttgctgg ccagcttacc tcccggtggt gggtcggtgg
     3421 tccctgggca ggggtctcca aatcccggac gagcccccaa atgaaagacc cccgtcgtgg
     3481 gtagtcaatc actcagagga gaccctccca aggaacagcg agaccacgat tcggatgcaa
     3541 acagcaagag gctttattgg gaatacgggt acccgggcga cgcagtctat cggaagactg
     3601 gcgcgccgag tgaggggttg tgggctcttt tattgagctc tgcttatata gacctcccac
     3661 cgtacacgcc taccgcccat ttgcgtcaat ggggcggagt tgttacgaca ttttggaaag
     3721 tcccgttgat tttggtgcca aaacaaactc ccattgacgt caatggggtg gagacttgga
     3781 aatccccgtg agtcaaaccg ctatccacgc ccattgatgt actgccaaaa ccgcatcacc
     3841 atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc ccataaggtc
     3901 atgtactggg cataatgcca ggcgggccat ttaccgtcat tgacgtcaat agggggcgta
     3961 cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa atactccacc
     4021 cattgacgtc aatggaaagt ccctattggc gttactatgg gaacatacgt cattattgac
     4081 gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt aagttatgta
     4141 acgcggaact ccatatatgg gctatgaact aatgaccccg taattgatta ctattaataa
     4201 ctagtcaata atcaatgtca acatggcggt aatgttggac atgagccaat ataaatgtac
     4261 atattatgat atggatacaa cgtatgcaat ggccaagcta attcttgaag acgaaagggc
     4321 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     4381 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     4441 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     4501 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     4561 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     4621 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     4681 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     4741 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     4801 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     4861 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     4921 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     4981 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     5041 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     5101 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     5161 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     5221 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     5281 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     5341 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     5401 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     5461 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     5521 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     5581 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     5641 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     5701 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     5761 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     5821 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     5881 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     5941 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     6001 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     6061 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     6121 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     6181 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     6241 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     6301 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     6361 c
//
