LOCUS       sequence-375710-        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_gRNA2_dTet_mTurquoise2
FEATURES             Location/Qualifiers
     source          1..6361
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(34..363)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      48..183
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     348..368
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     primer_bind     504..526
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     LTR             629..1054
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     CDS             complement(join(1197..1910,1911..1913,1914..1916))
                     /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(1242..1263)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1591..1610
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1850..1871
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1913..1922
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1939..2150)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     promoter        complement(2290..2538)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2348..2367)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2518..2538)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(2554..2970)
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     complement(2570..2586)
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     complement(2602..2624)
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     misc_feature    3171..3370
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     LTR             3433..3608
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     promoter        complement(3609..3812)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3638..3658)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3813..4192
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(4292..4310)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4378..4482
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4483..4551,4552..5343)
                     /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(4701..4720)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5514..6102
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6003..6022
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6256..6273
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(join(6354..6361,1..12))
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gctctgatgc cgcatagtta agccagtaag ctttttgcaa aagcctaggc ctccaaaaaa
       61 gcctcctcac tacttctgga atagctcaga ggccgaggcg gcctcggcct ctgcataaat
      121 aaaaaaaatt agtcagccat ggggcggaga atgggcggaa ctgggcggag ttaggggcgg
      181 gatgggcgga gttaggggcg ggactatggt tgctgactaa ttgagatgca tgctttgcat
      241 acttctgcct gctggggagc ctggggactt tccacacctg gttgctgact aattgagatg
      301 catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc ctaactgaca
      361 cacattccac agccggatct acactccgct atcgctacgt gactgggtca tggctgcgcc
      421 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
      481 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
      541 accgaaacgc gcgaggcagc cagcttacct cccggtggtg ggtcggtggt ccctgggcag
      601 gggtctcccg atcccggacg agcccccaaa tgaaagaccc ccgctgacgg gtagtcaatc
      661 actcagagga gaccctccca aggaacagcg agaccacaag tcggatgcaa ctgcaagagg
      721 gtttattgga tacacgggta cccgggcgac tcagtcaatc ggaggactgg cgccccgagt
      781 gaggggttgt gggctctttt attgagctcg gggagcagaa gcgcgcgaac agaagcgaga
      841 agcgaactga ttggttagtt caaataaggc acagggtcat ttcaggtcct tggggcaccc
      901 tggaaacatc tgatggttct ctagggtcga ccctgttcca tctgttcctg accttgatct
      961 gaacttctct attctcagtt atgtattttt ccatgccttg caaaatggcg ttacttaagc
     1021 tagcttgcca aacctacagg tggggtcttt cattcccccc tttttctgga gactaaataa
     1081 aatcttttat tttatctatg gctcgtactc tataggcttc agctggtgat attgttgagt
     1141 caaaactaga gcctggacca ctgatatcct gtctttaaca aattggacta atcgatttac
     1201 ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg tcacgaactc cagcaggacc
     1261 atgtgatcgc gcttctcgtt ggggtctttg ctcagcttgg actgggtgct caggtagtgg
     1321 ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt tctgctggta gtggtcggcg
     1381 agctgcacgc cgccgtcctc gatgttgtgg cggatcttga agttggcctt gatgccgttc
     1441 ttctgcttgt cggcggtgat atagacgttg tcgctaaagt agttgtactc cagcttgtgc
     1501 cccaggatgt tgccgtcctc cttgaagtcg atgcccttca gctcgatgcg gttcaccagg
     1561 gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt tgccgtcgtc cttgaagaag
     1621 atggtgcgct cctggacgta gccttcgggc atggcggact tgaagaagtc gtgctgcttc
     1681 atgtggtcgg ggtagcgggc gaagcactgc acgccccagg acagggtggt cacgagggtg
     1741 ggccagggca cgggcagctt gccggtggtg cagatgaact tcagggtcag cttgccgtag
     1801 gtggcatcgc cctcgccctc gccggacacg ctgaacttgt ggccgtttac gtcgccgtcc
     1861 agctcgacca ggatgggcac caccccggtg aacagctcct cgcccttgct caccatggtg
     1921 gcgaccgcgt cacgacacct gtgttctggc ggcaaacccg ttgcgaaaaa gaacgttcac
     1981 ggcgactact gcacttatat acggttctcc cccaccctcg ggaaaaaggc ggagccagta
     2041 cacgacatca ctttcccagt ttaccccgcg ccaccttctc taggcaccgg ttcaattgcc
     2101 gacccctccc cccaacttct cggggactgt gggcgatgtg cgctctgccc actgacgggc
     2161 accggagcca ctcgagaaaa aaagcaccga ctcggtgcca ctttttcaag ttgataacgg
     2221 actagcctta tttaaacttg ctatgctgtt tccagcatag ctcttaaacg cggttgcctg
     2281 cgcgcttacg gtgtttcgtc ctttccacaa gatatataaa gccaagaaat cgaaatactt
     2341 tcaagttacg gtaagcatat gatagtccat tttaaaacat aattttaaaa ctgcaaacta
     2401 cccaagaaat tattactttc tacgtcacgt attttgtact aatatctttg tgtttacagt
     2461 caaattaatt ccaattatct ctctaacagc cttgtatcgt atatgcaaat atgaaggaat
     2521 catgggaaat aggccctcgg atccttcaat tccggcgcct agagaaggag tgagggctgg
     2581 ataaagggag gatcgaggcg gggtcgaacg aggaggttca agggggagag acggggcgga
     2641 tggaggaaga ggaggcggag gcttagggtg tacaaagggc ttgacccagg gaggggggtc
     2701 aaaagccaag gcttcccagg tcacgatgta ggggacctgg tctgggtgtc catgcgggcc
     2761 aggtgaaaag accttgatct taacctgggt gatgaggtct cggttaaagg tgccgtctcg
     2821 cggccatccg acgttaaagg ttggccattc tgcagagcag aaggtaaccc aacgtctctt
     2881 cttgacatct accgactggt tgtgagcgat ccgctcgaca tctttccagt gacctaaggt
     2941 caaacttaag ggagtggtaa cagtctggcc ctaattttca gacaaataca gaaacacagt
     3001 cagacagaga caacacagaa cgatgctgca gcgctgcagc agacaagacg cgcggcttcg
     3061 gttccaaacc gaaagcaaaa attcagacgg aggcgggaac tgttttaggt tctcgtctcc
     3121 taccagaacc acatatcctg acggggtcgg attccacatc gactcccttc ctcaggtcgg
     3181 gccacaaaaa cggcccccaa agtccctggg acgtctccca gggttgcggc cgggtgttca
     3241 gaactcgtca gttccaccac gggtccgcca gatacagagc tagttagcta actagtacag
     3301 acgcaggcgc ataacatcaa acatagacac tagacaatcg gacagacaca gataagttgc
     3361 tggccagctt acctcccggt ggtgggtcgg tggtccctgg gcaggggtct ccaaatcccg
     3421 gacgagcccc caaatgaaag acccccgtcg tgggtagtca atcactcaga ggagaccctc
     3481 ccaaggaaca gcgagaccac gattcggatg caaacagcaa gaggctttat tgggaatacg
     3541 ggtacccggg cgacgcagtc tatcggaaga ctggcgcgcc gagtgagggg ttgtgggctc
     3601 ttttattgag ctctgcttat atagacctcc caccgtacac gcctaccgcc catttgcgtc
     3661 aatggggcgg agttgttacg acattttgga aagtcccgtt gattttggtg ccaaaacaaa
     3721 ctcccattga cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa ccgctatcca
     3781 cgcccattga tgtactgcca aaaccgcatc accatggtaa tagcgatgac taatacgtag
     3841 atgtactgcc aagtaggaaa gtcccataag gtcatgtact gggcataatg ccaggcgggc
     3901 catttaccgt cattgacgtc aatagggggc gtacttggca tatgatacac ttgatgtact
     3961 gccaagtggg cagtttaccg taaatactcc acccattgac gtcaatggaa agtccctatt
     4021 ggcgttacta tgggaacata cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt
     4081 cagccaggcg ggccatttac cgtaagttat gtaacgcgga actccatata tgggctatga
     4141 actaatgacc ccgtaattga ttactattaa taactagtca ataatcaatg tcaacatggc
     4201 ggtaatgttg gacatgagcc aatataaatg tacatattat gatatggata caacgtatgc
     4261 aatggccaag ctaattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt
     4321 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     4381 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     4441 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     4501 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     4561 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     4621 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     4681 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa
     4741 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     4801 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     4861 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     4921 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     4981 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca
     5041 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     5101 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     5161 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     5221 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     5281 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     5341 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     5401 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     5461 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     5521 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     5581 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     5641 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     5701 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     5761 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     5821 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     5881 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     5941 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     6001 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     6061 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     6121 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     6181 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     6241 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     6301 tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc
     6361 t
//
