LOCUS       sequence-467827-        5601 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the MS2-VP64-P65-HSF1 under the control of human EF1a 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5601)
  AUTHORS   Wu Y, Zhong A, Evangelisti A, Sidharta M, Danwei H, Studer L, Zhou T
  TITLE     Leveraging CRISPR activation for rapid assessment of gene editing 
            products in human pluripotent stem cells.
  JOURNAL   Stem Cell Reports. 2025 Apr 25:102499. doi: 
            10.1016/j.stemcr.2025.102499.
  PUBMED    40345204
REFERENCE   2  (bases 1 to 5601)
  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: "Stem Cell 
            Reports. 2025 Apr 25:102499. doi: 10.1016/j.stemcr.2025.102499."
COMMENT     Sequence Label: EF1a-MVPH
FEATURES             Location/Qualifiers
     source          1..5601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        82..1260
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          313..1251
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1208..1228
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1267..1276
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1276..1662
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /label=MS2-N55K
                     /note="binds with high affinity to a specific stem-loop 
                     structure in the viral RNA (Lim et al., 1994)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     CDS             1717..1737
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1762..1911
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             1939..2481
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             2506..2877
                     /codon_start=1
                     /product="C-terminal activation domain from the human heat 
                     shock transcription factor HSF1"
                     /label=HSF1
                     /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR
                     PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED
                     PTISLLTGSEPPKAKDPTVS"
     primer_bind     complement(2900..2917)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2906..3130
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3201..3217)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3201..3217)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3214..3236)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3225..3241
                     /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(3249..3255,3256..3273,3274..3279))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3294..3315
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3432..3449)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3603..4191)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3683..3702)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4362..5153,5154..5222))
                     /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     4985..5004
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5223..5327)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5406..5425)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctgcaaagat ggataaagtt ttaaacagag aggaatcttt gcagctaatg gaccttctag
       61 gtcttgaaag gagtgggaat tggctccggt gcccgtcagt gggcagagcg cacatcgccc
      121 acagtccccg agaagttggg gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg
      181 cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg
      241 ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc
      301 gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat
      361 ggcccttgcg tgccttgaat tacttccacc tggctgcagt acgtgattct tgatcccgag
      421 cttcgggttg gaagtgggtg ggagagttcg aggccttgcg cttaaggagc cccttcgcct
      481 cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac
      541 cttcgcgcct gtctcgctgc tttcgataag tctctagcca tttaaaattt ttgatgacct
      601 gctgcgacgc tttttttctg gcaagatagt cttgtaaatg cgggccaaga tctgcacact
      661 ggtatttcgg tttttggggc cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt
      721 tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg gacgggggta gtctcaagct
      781 ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca
      841 aggctggccc ggtcggcacc agttgcgtga gcggaaagat ggccgcttcc cggccctgct
      901 gcagggagct caaaatggag gacgcggcgc tcgggagagc gggcgggtga gtcacccaca
      961 caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg
     1021 gcgccgtcca ggcacctcga ttagttctcg agcttttgga gtacgtcgtc tttaggttgg
     1081 ggggaggggt tttatgcgat ggagtttccc cacactgagt gggtggagac tgaagttagg
     1141 ccagcttggc acttgatgta attctccttg gaatttgccc tttttgagtt tggatcttgg
     1201 ttcattctca agcctcagac agtggttcaa agtttttttc ttccatttca ggtgtcgtga
     1261 cgtacggcca ccatggcttc aaactttact cagttcgtgc tcgtggacaa tggtgggaca
     1321 ggggatgtga cagtggctcc ttctaatttc gctaatgggg tggcagagtg gatcagctcc
     1381 aactcacgga gccaggccta caaggtgaca tgcagcgtca ggcagtctag tgcccagaag
     1441 agaaagtata ccatcaaggt ggaggtcccc aaagtggcta cccagacagt gggcggagtc
     1501 gaactgcctg tcgccgcttg gaggtcctac ctgaacatgg agctcactat cccaattttc
     1561 gctaccaatt ctgactgtga actcatcgtg aaggcaatgc aggggctcct caaagacggt
     1621 aatcctatcc cttccgccat cgccgctaac tcaggtatct acagcgctgg aggaggtgga
     1681 agcggaggag gaggaagcgg aggaggaggt agcggaccta agaaaaagag gaaggtggcg
     1741 gccgctggat ccggacgggc tgacgcattg gacgattttg atctggatat gctgggaagt
     1801 gacgccctcg atgattttga ccttgacatg cttggttcgg atgcccttga tgactttgac
     1861 ctcgacatgc tcggcagtga cgcccttgat gatttcgacc tggacatgct gattaactgt
     1921 acaggcagtg gaggatcccc ttcagggcag atcagcaacc aggccctggc tctggcccct
     1981 agctccgctc cagtgctggc ccagactatg gtgccctcta gtgctatggt gcctctggcc
     2041 cagccacctg ctccagcccc tgtgctgacc ccaggaccac cccagtcact gagcgctcca
     2101 gtgcccaagt ctacacaggc cggcgagggg actctgagtg aagctctgct gcacctgcag
     2161 ttcgacgctg atgaggacct gggagctctg ctggggaaca gcaccgatcc cggagtgttc
     2221 acagatctgg cctccgtgga caactctgag tttcagcagc tgctgaatca gggcgtgtcc
     2281 atgtctcata gtacagccga accaatgctg atggagtacc ccgaagccat tacccggctg
     2341 gtgaccggca gccagcggcc ccccgacccc gctccaactc ccctgggaac cagcggcctg
     2401 cctaatgggc tgtccggaga tgaagacttc tcaagcatcg ctgatatgga ctttagtgcc
     2461 ctgctgtcac agatttcctc tagtgggcag ggaggaggtg gaagcggctt cagcgtggac
     2521 accagtgccc tgctggacct gttcagcccc tcggtgaccg tgcccgacat gagcctgcct
     2581 gaccttgaca gcagcctggc cagtatccaa gagctcctgt ctccccagga gccccccagg
     2641 cctcccgagg cagagaacag cagcccggat tcagggaagc agctggtgca ctacacagcg
     2701 cagccgctgt tcctgctgga ccccggctcc gtggacaccg ggagcaacga cctgccggtg
     2761 ctgtttgagc tgggagaggg ctcctacttc tccgaagggg acggcttcgc cgaggacccc
     2821 accatctccc tgctgacagg ctcggagcct cccaaagcca aggaccccac tgtctcctga
     2881 gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     2941 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     3001 aatgagaaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     3061 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     3121 ggctctatgg cttctgaggc ggaaagaacc agctggggct cgataccgtc gacctctagc
     3181 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     3241 attccacaca acatacgagc cggaagcata aagtgtaaag cctagggtgc ctaatgagtg
     3301 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     3361 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     3421 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     3481 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3541 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     3601 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3661 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     3721 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     3781 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     3841 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     3901 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     3961 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4021 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     4081 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4141 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4201 ttgatctttt ctacggggtc tgacactcag tggaacgaaa actcacgtta agggattttg
     4261 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     4321 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4381 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     4441 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     4501 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     4561 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     4621 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     4681 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     4741 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     4801 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     4861 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     4921 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     4981 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     5041 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     5101 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     5161 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     5221 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     5281 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     5341 aaagtgccac ctgacgtcga cggatcggga gatcgatctc ccgatcccct agggtcgact
     5401 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg
     5461 ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc
     5521 gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg
     5581 gccagatata cgcgttgcta g
//
