LOCUS       sequence-240450-        7819 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral overexpression of Bhlhe40 with a 3xFLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7819)
  AUTHORS   Henriksson J, Chen X, Gomes T, Ullah U, Meyer KB, Miragaia R, Duddy 
            G, Pramanik J, Yusa K, Lahesmaa R, Teichmann SA
  TITLE     Genome-wide CRISPR Screens in T Helper Cells Reveal Pervasive 
            Crosstalk between Activation and Differentiation.
  JOURNAL   Cell. 2019 Jan 7. pii: S0092-8674(18)31569-1. doi: 
            10.1016/j.cell.2018.11.044.
  PUBMED    30639098
REFERENCE   2  (bases 1 to 7819)
  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: "Cell. 2019 
            Jan 7. pii: S0092-8674(18)31569-1. doi: 10.1016/j.cell.2018.11.044."
COMMENT     Sequence Label: MSCV-Bhlhe40-3xFLAG-IRES-BFP
FEATURES             Location/Qualifiers
     source          1..7819
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..517
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    581..922
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             989..1405
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1335..1357
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1373..1389
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             1445..1510
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYEDDDDK"
     misc_feature    2782..3331
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2926..2943)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3153..3172
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3334..3336,3337..3339,3340..4053)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(3379..3400)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     LTR             4214..4728
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4890..4906
                     /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(4914..4920,4921..4938,4939..4944))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4959..4980
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5097..5114)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5268..5856)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5348..5367)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6027..6818,6819..6887))
                     /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     6650..6669
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6888..6992)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7060..7078
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7116..7138)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7238..7257
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7451..7473
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7601..7620
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc
       61 atggaaaata cataactgag aatagagaag ttcagatcaa ggttaggaac agagagacag
      121 cagaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
      181 gaacagatgg tccccagatg cggtcccgcc ctcagcagtt tctagagaac catcagatgt
      241 ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt
      301 tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac
      361 ccctcactcg gcgcgccagt cctccgatag actgcgtcgc ccgggtaccc gtattcccaa
      421 taaagcctct tgctgtttgc atccgaatcg tggactcgct gatccttggg agggtctcct
      481 cagattgatt gactgcccac ctcgggggtc tttcatttgg aggttccacc gagatttgga
      541 gacccctgcc cagggaccac cgaccccccc gccgggaggt aagctggcca gcggtcgttt
      601 cgtgtctgtc tctgtctttg tgcgtgtttg tgccggcatc taatgtttgc gcctgcgtct
      661 gtactagtta gctaactagc tctgtatctg gcggacccgt ggtggaactg acgagttctg
      721 aacacccggc cgcaaccctg ggagacgtcc cagggacttt gggggccgtt tttgtggccc
      781 gacctgagga agggagtcga tgtggaatcc gaccccgtca ggatatgtgg ttctggtagg
      841 agacgagaac ctaaaacagt tcccgcctcc gtctgaattt ttgctttcgg tttggaaccg
      901 aagccgcgcg tcttgtctgc tgcagcgctg cagcatcgtt ctgtgttgtc tctgtctgac
      961 tgtgtttctg tatttgtctg aaaattaggg ccagactgtt accactccct taagtttgac
     1021 cttaggtcac tggaaagatg tcgagcggat cgctcacaac cagtcggtag atgtcaagaa
     1081 gagacgttgg gttaccttct gctctgcaga atggccaacc tttaacgtcg gatggccgcg
     1141 agacggcacc tttaaccgag acctcatcac ccaggttaag atcaaggtct tttcacctgg
     1201 cccgcatgga cacccagacc aggtccccta catcgtgacc tgggaagcct tggcttttga
     1261 cccccctccc tgggtcaagc cctttgtaca ccctaagcct ccgcctcctc ttcctccatc
     1321 cgccccgtct ctcccccttg aacctcctcg ttcgaccccg cctcgatcct ccctttatcc
     1381 agccctcact ccttctctag gcgccggaat tcagatctta cgtagctagc ggatcttaac
     1441 catggactac aaagaccatg acggtgatta taaagatcat gacatcgatt acgaggatga
     1501 cgatgacaag gaggaacgga tccccagcgc gcaaccacct cctacctgcc tgcccaaagc
     1561 tccagggctg gagcacggag acctgtcagg gatggatttt gcccacatgt accaagtgta
     1621 caagtccagg cggggaataa aacggagcga agacagcaag gaaacttaca aactgccgca
     1681 ccggctgatt gagaaaaaga gacgtgaccg gattaacgag tgcattgccc agctgaagga
     1741 tctcctaccc gaacatctca aacttactac tttgggtcac ttggaaaaag cagtggttct
     1801 ggagcttacg ttgaagcacg tgaaagcatt gacaaatcta attgatcagc agcagcagaa
     1861 aatcattgcc ctgcagagcg gtttacaagc tggtgatttg tcgggaagaa atctcgaggc
     1921 agggcaagaa atgttctgct caggtttcca gacttgtgcc cgtgaggtac ttcagtacct
     1981 ggcgaagcat gagaacactc gggacctgaa atcttcccag ctcgtcactc atctccatcg
     2041 tgtggtctcg gagctgctgc agggtggtgc ttccaggaaa ccattggact cggctcccaa
     2101 agccgtggac ttgaaagaga agcccagctt cctagccaag ggatcagaag gccctgggaa
     2161 aaactgtgtg ccagtcatcc agcggacttt tgctccctcg ggtggggagc agagcggcag
     2221 tgacacggac acagacagtg gctatggagg tgaattggag aagggggact tgcgcagtga
     2281 gcagccgtac ttcaaaagcg accatggacg caggttcgcc gtgggagaac gtgtcagcac
     2341 aattaagcaa gaatccgaag agccccccac caaaaagagc cgaatgcagc tctcagaaga
     2401 ggaaggccac ttcgcgggca gtgatctgat gggttcccca tttcttgggc cacacccaca
     2461 tcagcctcct ttttgccttc ccttctatct catcccacca tcggccactg cctacctgcc
     2521 tatgctggag aaatgctggt accccacctc tgtgccagtg ttatacccag gcctcaacac
     2581 ctcagctgca gccctctcca gcttcatgaa cccagacaag ataccgactc ccttgcttct
     2641 gccccagaga ctcccttctc ctttggcaca ttcgtccctc gactcttcgg ccttgctcca
     2701 ggctttgaag cagatccctc ctttaaactt agaaaccaaa gactaatagg ctcgagcggg
     2761 atcaattccg ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg
     2821 cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga
     2881 aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa
     2941 tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa
     3001 caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct
     3061 gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg
     3121 ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg
     3181 ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca
     3241 catgctttac atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg
     3301 acgtggtttt cctttgaaaa acacgataat accatggtga gcaagggcga ggagctgttc
     3361 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     3421 gtgaggggcg agggcgaggg cgatgccacc aacggcaagc tgaccctgaa gttcatctgc
     3481 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgag ccacggcgtg
     3541 cagtgcttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     3601 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcac ctacaagacc
     3661 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcgtc
     3721 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaactt caacagccac
     3781 aacatctata tcatggccgt caagcagaag aacggcatca aggtgaactt caagatccgc
     3841 cacaacgtgg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     3901 ggcgacggcc ccgtgctgct gcccgacagc cactacctga gcacccagtc cgtgctgagc
     3961 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttccgcac cgccgccggg
     4021 atcactctcg gcatggacga gctgtacaag taagcggccg cgactctaga gtcgacctgc
     4081 aggcatgcaa gcttcaggta gccggctaac gttaacaacc ggtacctcta gaactatagc
     4141 tagcatgcgc aaatttaaag cgctgatatc gataaaataa aagattttat ttagtctcca
     4201 gaaaaagggg ggaatgaaag accccacctg taggtttggc aagctagctt aagtaacgcc
     4261 attttgcaag gcatggaaaa tacataactg agaatagaga agttcagatc aaggttagga
     4321 acagagagac agcagaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg
     4381 gctcagggcc aagaacagat ggtccccaga tgcggtcccg ccctcagcag tttctagaga
     4441 accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac
     4501 taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa
     4561 agagcccaca acccctcact cggcgcgcca gtcctccgat agactgcgtc gcccgggtac
     4621 ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc tgttccttgg
     4681 gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcatgg gtaacagttt
     4741 cttgaagttg gagaacaaca ttctgagggt aggagtcgaa tattaagtaa tcctgactca
     4801 attagccact gttttgaatc cacatactcc aatactcctg aaatagttca ttatggacag
     4861 cgcagaaaga gctggggaga attgtgaaat tgttatccgc tcacaattcc acacaacata
     4921 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     4981 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     5041 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     5101 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     5161 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     5221 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     5281 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     5341 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     5401 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     5461 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     5521 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     5581 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     5641 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     5701 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     5761 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     5821 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     5881 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     5941 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     6001 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     6061 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     6121 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     6181 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     6241 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     6301 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     6361 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     6421 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     6481 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     6541 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     6601 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     6661 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     6721 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     6781 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     6841 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     6901 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     6961 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     7021 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     7081 tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     7141 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     7201 gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg
     7261 agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     7321 aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
     7381 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
     7441 ccagggtttt cccagtcacg acgttgtaaa acgacggcgc aaggaatggt gcatgcaagg
     7501 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
     7561 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
     7621 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagaggc
     7681 gattagtcca atttgttaaa gacaggatat cagtggtcca ggctctagtt ttgactcaac
     7741 aatatcacca gctgaagcct atagagtacg agccatagat aaaataaaag attttattta
     7801 gtctccagaa aaagggggg
//
