LOCUS       sequence-493702-        4942 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 4942)
  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 4942)
  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: pPD0054_pIVTRup_mCherry2
FEATURES             Location/Qualifiers
     source          1..4942
                     /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"
     primer_bind     complement(377..395)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(1248..1264)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1248..1264)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1261..1283)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1272..1288
                     /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(1296..1302,1303..1320,1321..1326))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1341..1362
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1479..1496)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1650..2238)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1730..1749)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2409..3200,3201..3269))
                     /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     3032..3051
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(3287..4081)
                     /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(3398..3417)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4008..4027
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      complement(4425..4853)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4535..4556)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4747..4766
                     /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 tatggtgagc
      241 aagggcgagg aggacaacat ggccatcatc aaggagttca tgaggttcaa ggtgcacatg
      301 gagggcagcg tgaacggcca cgagttcgag atcgagggcg agggcgaggg caggccctac
      361 gagggcaccc agaccgccaa gctgaaggtg accaagggcg gccccctgcc cttcgcctgg
      421 gacatcctga gcccccagtt catgtacggc agcaaggcct acgtgaagca ccccgccgac
      481 atccccgact acctgaagct gagcttcccc gagggcttca actgggagag ggtgatgaac
      541 ttcgaggacg gcggcgtggt gaccgtgacc caggacagca gcctgcagga cggcgagttc
      601 atctacaagg tgaagctgag gggcaccaac ttccccagcg acggccccgt gatgcagtgc
      661 aggaccatgg gctgggaggc cagcaccgag aggatgtacc ccgaggacgg cgccctgaag
      721 ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc cgaggtgaag
      781 accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt ggacatcaag
      841 ctggacatcc tgagccacaa cgaggactac accatcgtgg agcagtacga gagggccgag
      901 ggcaggcaca gcaccggcgg catggacgag ctgtacaagt gaatcgctgc cttctgcggg
      961 gcttgccttc tggccatgcc cttcttctct cccttgcacc tgtacctctt ggtctttgaa
     1021 taaagcctga gtaggaagtg agggtctaga aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     1081 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaacgatcaa
     1141 taaccgacac tcctgcgtcg cctacgcgct acgatctgca gaattccagc acactggcgg
     1201 ccgttactag tggatccgag ctcggtacca agcttggcgt aatcatggtc atagctgttt
     1261 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     1321 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     1381 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     1441 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     1501 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     1561 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     1621 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     1681 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     1741 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     1801 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     1861 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     1921 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     1981 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     2041 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     2101 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     2161 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     2221 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     2281 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     2341 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     2401 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     2461 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     2521 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     2581 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     2641 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     2701 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     2761 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     2821 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     2881 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     2941 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     3001 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     3061 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     3121 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     3181 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     3241 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaattcag aagaactcgt
     3301 caagaaggcg atagaaggcg atgcgctgcg aatcgggagc ggcgataccg taaagcacga
     3361 ggaagcggtc agcccattcg ccgccaagct cttcagcaat atcacgggta gccaacgcta
     3421 tgtcctgata gcggtccgcc acacccagcc ggccacagtc gatgaatcca gaaaagcggc
     3481 cattttccac catgatattc ggcaagcagg catcgccatg ggtcacgacg agatcctcgc
     3541 cgtcgggcat gcgcgccttg agcctggcga acagttcggc tggcgcgagc ccctgatgct
     3601 cttcgtccag atcatcctga tcgacaagac cggcttccat ccgagtacgt gctcgctcga
     3661 tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc
     3721 gcattgcatc agccatgatg gatactttct cggcaggagc aaggtgggat gacaggagat
     3781 cctgccccgg cacttcgccc aatagcagcc agtcccttcc cgcttcagtg acaacgtcga
     3841 gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct
     3901 gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg
     3961 ctgacagccg gaacacggcg gcatcagagc agccgattgt ctgttgtgcc cagtcatagc
     4021 cgaatagcct ctccacccaa gcggccggag aacctgcgtg caatccatct tgttcaatca
     4081 tgcgaaacga tcctcatcct gtctcttgat cagatcttga tcccctgcgc catcagatcc
     4141 ttggcggcaa gaaagccatc cagtttactt tgcagggctt cccaacctta ccagagggcg
     4201 ccccagctgg caattccggt tcgcttgctg tccataaaac cgcccagtct agctatcgcc
     4261 atgtaagccc actgcaagct acctgctttc tctttgcgct tgcgttttcc cttgtccaga
     4321 tagcccagta gctgacattc atccggggtc agcaccgttt ctgcggactg gctttctacg
     4381 tgttccgctt cctttagcag cccttgcgcc ctgaattttg ttaaaattcg cgttaaattt
     4441 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     4501 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     4561 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     4621 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     4681 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     4741 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     4801 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtccattcg
     4861 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     4921 cagctggcga aagggggatg tg
//
