LOCUS       sequence-493699-        4626 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 4626)
  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 4626)
  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: pEP0016_pIVTRup(BsmBI)_BSD
FEATURES             Location/Qualifiers
     source          1..4626
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     127..149
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     141..158
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     142..158
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        165..183
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        255..273
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      313..322
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             319..717
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(1022..1038)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1022..1038)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1035..1057)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1046..1062
                     /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(1070..1076,1077..1094,1095..1100))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1115..1136
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1253..1270)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1424..2012)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1504..1523)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2183..2974,2975..3043))
                     /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     2806..2825
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(3061..3855)
                     /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(3172..3191)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3782..3801
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      complement(join(4199..4626,1))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4309..4330)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4521..4540
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
       61 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
      121 ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgtaatac gactcactat
      181 agggcgaatt gggccctcta gatgcatgct cgagcggccg ccagtgtgat ggatttggac
      241 cctcgtacag aagctaatac gactcactat agggaaataa gagagaaaag aagagtaaga
      301 agaaatataa gagccaccat ggccaagcct ttgtctcaag aagaatccac cctcattgaa
      361 agagcaacgg ctacaatcaa cagcatcccc atctctgaag actacagcgt cgccagcgca
      421 gctctctcta gcgacggccg catcttcact ggtgtcaatg tatatcattt tactggggga
      481 ccttgtgcag aactcgtggt gctgggcact gctgctgctg cggcagctgg caacctgact
      541 tgtatcgtcg cgatcggaaa tgagaacagg ggcatcttga gcccctgcgg acggtgccga
      601 caggtgcttc tcgatctgca tcctgggatc aaagccatag tgaaggacag tgatggacag
      661 ccgacggcag ttgggattcg tgaattgctg ccctctggtt atgtgtggga gggctaaatc
      721 gctgccttct gcggggcttg ccttctggcc atgcccttct tctctccctt gcacctgtac
      781 ctcttggtct ttgaataaag cctgagtagg aagtgagggt ctagaaaaaa aaaaaaaaaa
      841 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
      901 tgagacgcga tcaataaccg acactcctgc gtcgcctacg cgctacgatc tgcagaattc
      961 cagcacactg gcggccgtta ctagtggatc cgagctcggt accaagcttg gcgtaatcat
     1021 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     1081 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     1141 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     1201 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     1261 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     1321 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     1381 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     1441 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     1501 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     1561 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     1621 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     1681 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     1741 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     1801 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     1861 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     1921 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     1981 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     2041 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     2101 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     2161 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     2221 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     2281 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     2341 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     2401 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     2461 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     2521 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     2581 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     2641 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     2701 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     2761 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     2821 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     2881 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     2941 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     3001 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     3061 tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat
     3121 accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg
     3181 ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa
     3241 tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac
     3301 gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc
     3361 gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt
     3421 acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag
     3481 cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg
     3541 ggatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc
     3601 agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg
     3661 cgctgcctcg tcctgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac
     3721 cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg
     3781 tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc
     3841 atcttgttca atcatgcgaa acgatcctca tcctgtctct tgatcagatc ttgatcccct
     3901 gcgccatcag atccttggcg gcaagaaagc catccagttt actttgcagg gcttcccaac
     3961 cttaccagag ggcgccccag ctggcaattc cggttcgctt gctgtccata aaaccgccca
     4021 gtctagctat cgccatgtaa gcccactgca agctacctgc tttctctttg cgcttgcgtt
     4081 ttcccttgtc cagatagccc agtagctgac attcatccgg ggtcagcacc gtttctgcgg
     4141 actggctttc tacgtgttcc gcttccttta gcagcccttg cgccctgaat tttgttaaaa
     4201 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     4261 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     4321 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     4381 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     4441 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     4501 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     4561 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     4621 ggcgcg
//
