LOCUS       sequence-375703-        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_gRNA1_dTet_mTurquoise2
FEATURES             Location/Qualifiers
     source          1..6361
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     213..233
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     primer_bind     369..391
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     LTR             494..919
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     CDS             complement(join(1062..1775,1776..1778,1779..1781))
                     /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(1107..1128)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1456..1475
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1715..1736
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1778..1787
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1804..2015)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     promoter        complement(2155..2403)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2213..2232)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2383..2403)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(2419..2835)
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     complement(2435..2451)
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     complement(2467..2489)
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     misc_feature    3036..3235
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     LTR             3298..3473
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     promoter        complement(3474..3677)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3503..3523)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3678..4057
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(4157..4175)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4243..4347
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4348..4416,4417..5208)
                     /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(4566..4585)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5379..5967
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5868..5887
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6121..6138
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(6219..6238)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        complement(join(6260..6361,1..228))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(6274..6361,1..48)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 gccatggggc ggagaatggg cggaactggg cggagttagg ggcgggatgg gcggagttag
       61 gggcgggact atggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg
      121 ggagcctggg gactttccac acctggttgc tgactaattg agatgcatgc tttgcatact
      181 tctgcctgct ggggagcctg gggactttcc acaccctaac tgacacacat tccacagccg
      241 gatctacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
      301 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
      361 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
      421 gcagccagct tacctcccgg tggtgggtcg gtggtccctg ggcaggggtc tcccgatccc
      481 ggacgagccc ccaaatgaaa gacccccgct gacgggtagt caatcactca gaggagaccc
      541 tcccaaggaa cagcgagacc acaagtcgga tgcaactgca agagggttta ttggatacac
      601 gggtacccgg gcgactcagt caatcggagg actggcgccc cgagtgaggg gttgtgggct
      661 cttttattga gctcggggag cagaagcgcg cgaacagaag cgagaagcga actgattggt
      721 tagttcaaat aaggcacagg gtcatttcag gtccttgggg caccctggaa acatctgatg
      781 gttctctagg gtcgaccctg ttccatctgt tcctgacctt gatctgaact tctctattct
      841 cagttatgta tttttccatg ccttgcaaaa tggcgttact taagctagct tgccaaacct
      901 acaggtgggg tctttcattc cccccttttt ctggagacta aataaaatct tttattttat
      961 ctatggctcg tactctatag gcttcagctg gtgatattgt tgagtcaaaa ctagagcctg
     1021 gaccactgat atcctgtctt taacaaattg gactaatcga tttacttgta cagctcgtcc
     1081 atgccgagag tgatcccggc ggcggtcacg aactccagca ggaccatgtg atcgcgcttc
     1141 tcgttggggt ctttgctcag cttggactgg gtgctcaggt agtggttgtc gggcagcagc
     1201 acggggccgt cgccgatggg ggtgttctgc tggtagtggt cggcgagctg cacgccgccg
     1261 tcctcgatgt tgtggcggat cttgaagttg gccttgatgc cgttcttctg cttgtcggcg
     1321 gtgatataga cgttgtcgct aaagtagttg tactccagct tgtgccccag gatgttgccg
     1381 tcctccttga agtcgatgcc cttcagctcg atgcggttca ccagggtgtc gccctcgaac
     1441 ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga agaagatggt gcgctcctgg
     1501 acgtagcctt cgggcatggc ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag
     1561 cgggcgaagc actgcacgcc ccaggacagg gtggtcacga gggtgggcca gggcacgggc
     1621 agcttgccgg tggtgcagat gaacttcagg gtcagcttgc cgtaggtggc atcgccctcg
     1681 ccctcgccgg acacgctgaa cttgtggccg tttacgtcgc cgtccagctc gaccaggatg
     1741 ggcaccaccc cggtgaacag ctcctcgccc ttgctcacca tggtggcgac cgcgtcacga
     1801 cacctgtgtt ctggcggcaa acccgttgcg aaaaagaacg ttcacggcga ctactgcact
     1861 tatatacggt tctcccccac cctcgggaaa aaggcggagc cagtacacga catcactttc
     1921 ccagtttacc ccgcgccacc ttctctaggc accggttcaa ttgccgaccc ctccccccaa
     1981 cttctcgggg actgtgggcg atgtgcgctc tgcccactga cgggcaccgg agccactcga
     2041 gaaaaaaagc accgactcgg tgccactttt tcaagttgat aacggactag ccttatttaa
     2101 acttgctatg ctgtttccag catagctctt aaaccggcat gcgtattccc gtacggtgtt
     2161 tcgtcctttc cacaagatat ataaagccaa gaaatcgaaa tactttcaag ttacggtaag
     2221 catatgatag tccattttaa aacataattt taaaactgca aactacccaa gaaattatta
     2281 ctttctacgt cacgtatttt gtactaatat ctttgtgttt acagtcaaat taattccaat
     2341 tatctctcta acagccttgt atcgtatatg caaatatgaa ggaatcatgg gaaataggcc
     2401 ctcggatcct tcaattccgg cgcctagaga aggagtgagg gctggataaa gggaggatcg
     2461 aggcggggtc gaacgaggag gttcaagggg gagagacggg gcggatggag gaagaggagg
     2521 cggaggctta gggtgtacaa agggcttgac ccagggaggg gggtcaaaag ccaaggcttc
     2581 ccaggtcacg atgtagggga cctggtctgg gtgtccatgc gggccaggtg aaaagacctt
     2641 gatcttaacc tgggtgatga ggtctcggtt aaaggtgccg tctcgcggcc atccgacgtt
     2701 aaaggttggc cattctgcag agcagaaggt aacccaacgt ctcttcttga catctaccga
     2761 ctggttgtga gcgatccgct cgacatcttt ccagtgacct aaggtcaaac ttaagggagt
     2821 ggtaacagtc tggccctaat tttcagacaa atacagaaac acagtcagac agagacaaca
     2881 cagaacgatg ctgcagcgct gcagcagaca agacgcgcgg cttcggttcc aaaccgaaag
     2941 caaaaattca gacggaggcg ggaactgttt taggttctcg tctcctacca gaaccacata
     3001 tcctgacggg gtcggattcc acatcgactc ccttcctcag gtcgggccac aaaaacggcc
     3061 cccaaagtcc ctgggacgtc tcccagggtt gcggccgggt gttcagaact cgtcagttcc
     3121 accacgggtc cgccagatac agagctagtt agctaactag tacagacgca ggcgcataac
     3181 atcaaacata gacactagac aatcggacag acacagataa gttgctggcc agcttacctc
     3241 ccggtggtgg gtcggtggtc cctgggcagg ggtctccaaa tcccggacga gcccccaaat
     3301 gaaagacccc cgtcgtgggt agtcaatcac tcagaggaga ccctcccaag gaacagcgag
     3361 accacgattc ggatgcaaac agcaagaggc tttattggga atacgggtac ccgggcgacg
     3421 cagtctatcg gaagactggc gcgccgagtg aggggttgtg ggctctttta ttgagctctg
     3481 cttatataga cctcccaccg tacacgccta ccgcccattt gcgtcaatgg ggcggagttg
     3541 ttacgacatt ttggaaagtc ccgttgattt tggtgccaaa acaaactccc attgacgtca
     3601 atggggtgga gacttggaaa tccccgtgag tcaaaccgct atccacgccc attgatgtac
     3661 tgccaaaacc gcatcaccat ggtaatagcg atgactaata cgtagatgta ctgccaagta
     3721 ggaaagtccc ataaggtcat gtactgggca taatgccagg cgggccattt accgtcattg
     3781 acgtcaatag ggggcgtact tggcatatga tacacttgat gtactgccaa gtgggcagtt
     3841 taccgtaaat actccaccca ttgacgtcaa tggaaagtcc ctattggcgt tactatggga
     3901 acatacgtca ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc aggcgggcca
     3961 tttaccgtaa gttatgtaac gcggaactcc atatatgggc tatgaactaa tgaccccgta
     4021 attgattact attaataact agtcaataat caatgtcaac atggcggtaa tgttggacat
     4081 gagccaatat aaatgtacat attatgatat ggatacaacg tatgcaatgg ccaagctaat
     4141 tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     4201 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     4261 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     4321 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     4381 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     4441 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     4501 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     4561 gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc
     4621 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     4681 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     4741 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     4801 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     4861 ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta
     4921 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     4981 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     5041 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     5101 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     5161 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     5221 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5281 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5341 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5401 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5461 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5521 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5581 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5641 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5701 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5761 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5821 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     5881 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     5941 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6001 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     6061 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     6121 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     6181 ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca
     6241 tagttaagcc agtaagcttt ttgcaaaagc ctaggcctcc aaaaaagcct cctcactact
     6301 tctggaatag ctcagaggcc gaggcggcct cggcctctgc ataaataaaa aaaattagtc
     6361 a
//
