LOCUS       sequence-493693-        5649 bp    DNA     circular SYN 21-NOV-2025
DEFINITION  IVT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5649)
  AUTHORS   Datlinger P, Pankevich EV, Arnold CD, Pranckevicius N, Lin J, 
            Romanovskaia D, Schaefer M, Piras F, Orts AC, Nemc A, Biesaga PN, 
            Chan M, Neuwirth T, Artemov AV, Li W, Ladstatter S, Krausgruber T, 
            Bock C
  TITLE     Systematic discovery of CRISPR-boosted CAR T cell immunotherapies.
  JOURNAL   Nature. 2025 Oct;646(8086):963-972. doi: 10.1038/s41586-025-09507-9.
            Epub 2025 Sep 24.
  PUBMED    40993398
REFERENCE   2  (bases 1 to 5649)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 21, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41586-025-09507-9"; journalName: "Nature"; date: 
            "2025-10"; volume: "646"; issue: "8086"; pages: "963-972"
COMMENT     Sequence Label: pPD0055_pIVTRup_MS2-p65-HSF1
FEATURES             Location/Qualifiers
     source          1..5649
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     37..59
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     51..68
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     52..68
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        75..93
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        165..183
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             235..621
                     /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             676..696
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             712..1254
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             1279..1650
                     /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(1955..1971)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1955..1971)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1968..1990)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1979..1995
                     /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(2003..2009,2010..2027,2028..2033))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2048..2069
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2186..2203)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2357..2945)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2437..2456)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3116..3907,3908..3976))
                     /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     3739..3758
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(3994..4788)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4105..4124)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4715..4734
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      complement(5132..5560)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5242..5263)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5454..5473
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
       61 cggccagtga attgtaatac gactcactat agggcgaatt gggccctcta gatgcatgct
      121 cgagcggccg ccagtgtgat ggatttggac cctcgtacag aagctaatac gactcactat
      181 agggaaataa gagagaaaag aagagtaaga agaaatataa gagccaccga tatggcttca
      241 aactttactc agttcgtgct cgtggacaat ggtgggacag gggatgtgac agtggctcct
      301 tctaatttcg ctaatggggt ggcagagtgg atcagctcca actcacggag ccaggcctac
      361 aaggtgacat gcagcgtcag gcagtctagt gcccagaaga gaaagtatac catcaaggtg
      421 gaggtcccca aagtggctac ccagacagtg ggcggagtcg aactgcctgt cgccgcttgg
      481 aggtcctacc tgaacatgga gctcactatc ccaattttcg ctaccaattc tgactgtgaa
      541 ctcatcgtga aggcaatgca ggggctcctc aaagacggta atcctatccc ttccgccatc
      601 gccgctaact caggtatcta cagcgctgga ggaggtggaa gcggaggagg aggaagcgga
      661 ggaggaggta gcggacctaa gaaaaagagg aaggtggcgg ccgctggatc cccttcaggg
      721 cagatcagca accaggccct ggctctggcc cctagctccg ctccagtgct ggcccagact
      781 atggtgccct ctagtgctat ggtgcctctg gcccagccac ctgctccagc ccctgtgctg
      841 accccaggac caccccagtc actgagcgct ccagtgccca agtctacaca ggccggcgag
      901 gggactctga gtgaagctct gctgcacctg cagttcgacg ctgatgagga cctgggagct
      961 ctgctgggga acagcaccga tcccggagtg ttcacagatc tggcctccgt ggacaactct
     1021 gagtttcagc agctgctgaa tcagggcgtg tccatgtctc atagtacagc cgaaccaatg
     1081 ctgatggagt accccgaagc cattacccgg ctggtgaccg gcagccagcg gccccccgac
     1141 cccgctccaa ctcccctggg aaccagcggc ctgcctaatg ggctgtccgg agatgaagac
     1201 ttctcaagca tcgctgatat ggactttagt gccctgctgt cacagatttc ctctagtggg
     1261 cagggaggag gtggaagcgg cttcagcgtg gacaccagtg ccctgctgga cctgttcagc
     1321 ccctcggtga ccgtgcccga catgagcctg cctgaccttg acagcagcct ggccagtatc
     1381 caagagctcc tgtctcccca ggagcccccc aggcctcccg aggcagagaa cagcagcccg
     1441 gattcaggga agcagctggt gcactacaca gcgcagccgc tgttcctgct ggaccccggc
     1501 tccgtggaca ccgggagcaa cgacctgccg gtgctgtttg agctgggaga gggctcctac
     1561 ttctccgaag gggacggctt cgccgaggac cccaccatct ccctgctgac aggctcggag
     1621 cctcccaaag ccaaggaccc cactgtctcc tgtacatgaa tcgctgcctt ctgcggggct
     1681 tgccttctgg ccatgccctt cttctctccc ttgcacctgt acctcttggt ctttgaataa
     1741 agcctgagta ggaagtgagg gtctagaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     1801 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa cgatcaataa ccgacactcc
     1861 tgcgtcgcct acgcgctacg atctgcagaa ttccagcaca ctggcggccg ttactagtgg
     1921 atccgagctc ggtaccaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     1981 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     2041 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     2101 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     2161 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     2221 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     2281 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     2341 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     2401 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     2461 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     2521 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     2581 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     2641 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2701 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     2761 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     2821 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     2881 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     2941 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     3001 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     3061 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     3121 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     3181 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     3241 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     3301 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     3361 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     3421 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     3481 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     3541 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     3601 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     3661 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     3721 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     3781 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     3841 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     3901 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     3961 aatgttgaat actcatactc ttcctttttc aattcagaag aactcgtcaa gaaggcgata
     4021 gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa agcacgagga agcggtcagc
     4081 ccattcgccg ccaagctctt cagcaatatc acgggtagcc aacgctatgt cctgatagcg
     4141 gtccgccaca cccagccggc cacagtcgat gaatccagaa aagcggccat tttccaccat
     4201 gatattcggc aagcaggcat cgccatgggt cacgacgaga tcctcgccgt cgggcatgcg
     4261 cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt cgtccagatc
     4321 atcctgatcg acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc
     4381 ttggtggtcg aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc
     4441 catgatggat actttctcgg caggagcaag gtgggatgac aggagatcct gccccggcac
     4501 ttcgcccaat agcagccagt cccttcccgc ttcagtgaca acgtcgagca cagctgcgca
     4561 aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc tcgtcctgca gttcattcag
     4621 ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa
     4681 cacggcggca tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc
     4741 cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc
     4801 tcatcctgtc tcttgatcag atcttgatcc cctgcgccat cagatccttg gcggcaagaa
     4861 agccatccag tttactttgc agggcttccc aaccttacca gagggcgccc cagctggcaa
     4921 ttccggttcg cttgctgtcc ataaaaccgc ccagtctagc tatcgccatg taagcccact
     4981 gcaagctacc tgctttctct ttgcgcttgc gttttccctt gtccagatag cccagtagct
     5041 gacattcatc cggggtcagc accgtttctg cggactggct ttctacgtgt tccgcttcct
     5101 ttagcagccc ttgcgccctg aattttgtta aaattcgcgt taaatttttg ttaaatcagc
     5161 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     5221 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     5281 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     5341 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     5401 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     5461 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     5521 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc
     5581 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
     5641 ggggatgtg
//
