LOCUS       sequence-467830-        5415 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the MS2-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 5415)
  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 5415)
  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-MPH
FEATURES             Location/Qualifiers
     source          1..5415
                     /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             1753..2295
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             2320..2691
                     /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(2714..2731)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2720..2944
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3015..3031)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3015..3031)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3028..3050)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3039..3055
                     /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(3063..3069,3070..3087,3088..3093))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3108..3129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3246..3263)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3417..4005)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3497..3516)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4176..4967,4968..5036))
                     /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     4799..4818
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5037..5141)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5220..5239)
                     /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 ccccttcagg gcagatcagc aaccaggccc tggctctggc ccctagctcc
     1801 gctccagtgc tggcccagac tatggtgccc tctagtgcta tggtgcctct ggcccagcca
     1861 cctgctccag cccctgtgct gaccccagga ccaccccagt cactgagcgc tccagtgccc
     1921 aagtctacac aggccggcga ggggactctg agtgaagctc tgctgcacct gcagttcgac
     1981 gctgatgagg acctgggagc tctgctgggg aacagcaccg atcccggagt gttcacagat
     2041 ctggcctccg tggacaactc tgagtttcag cagctgctga atcagggcgt gtccatgtct
     2101 catagtacag ccgaaccaat gctgatggag taccccgaag ccattacccg gctggtgacc
     2161 ggcagccagc ggccccccga ccccgctcca actcccctgg gaaccagcgg cctgcctaat
     2221 gggctgtccg gagatgaaga cttctcaagc atcgctgata tggactttag tgccctgctg
     2281 tcacagattt cctctagtgg gcagggagga ggtggaagcg gcttcagcgt ggacaccagt
     2341 gccctgctgg acctgttcag cccctcggtg accgtgcccg acatgagcct gcctgacctt
     2401 gacagcagcc tggccagtat ccaagagctc ctgtctcccc aggagccccc caggcctccc
     2461 gaggcagaga acagcagccc ggattcaggg aagcagctgg tgcactacac agcgcagccg
     2521 ctgttcctgc tggaccccgg ctccgtggac accgggagca acgacctgcc ggtgctgttt
     2581 gagctgggag agggctccta cttctccgaa ggggacggct tcgccgagga ccccaccatc
     2641 tccctgctga caggctcgga gcctcccaaa gccaaggacc ccactgtctc ctgagtttaa
     2701 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     2761 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     2821 aaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     2881 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     2941 atggcttctg aggcggaaag aaccagctgg ggctcgatac cgtcgacctc tagctagagc
     3001 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3061 cacaacatac gagccggaag cataaagtgt aaagcctagg gtgcctaatg agtgagctaa
     3121 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3181 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     3241 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3301 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3361 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3421 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3481 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     3541 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3601 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     3661 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     3721 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     3781 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     3841 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     3901 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     3961 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4021 ttttctacgg ggtctgacac tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4081 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4141 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4201 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4261 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     4321 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     4381 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     4441 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     4501 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     4561 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     4621 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     4681 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     4741 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     4801 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     4861 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     4921 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     4981 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5041 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5101 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     5161 ccacctgacg tcgacggatc gggagatcga tctcccgatc ccctagggtc gactctcagt
     5221 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
     5281 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
     5341 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
     5401 tatacgcgtt gctag
//
