LOCUS       sequence-493701-        4952 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 4952)
  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 4952)
  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: pEP0001_pIVTRup(BsmBI)_EGFP
FEATURES             Location/Qualifiers
     source          1..4952
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(284..300)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(284..300)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(297..319)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    308..324
                     /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(332..338,339..356,357..362))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    377..398
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(515..532)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(686..1274)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(766..785)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1445..2236,2237..2305))
                     /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     2068..2087
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(2323..3117)
                     /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(2434..2453)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3044..3063
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      complement(3461..3889)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3571..3592)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3783..3802
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4015..4037
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4029..4046
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4030..4046
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        4053..4071
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        4143..4161
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4201..4210
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4207..4209,4210..4212,4213..4926)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4207..4209,4210..4212,4213..4923)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4252..4273)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4513..4532)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4860..4881
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 tctggccatg cccttcttct ctcccttgca cctgtacctc ttggtctttg aataaagcct
       61 gagtaggaag tgagggtcta gaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
      121 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aatgagacgc gatcaataac
      181 cgacactcct gcgtcgccta cgcgctacga tctgcagaat tccagcacac tggcggccgt
      241 tactagtgga tccgagctcg gtaccaagct tggcgtaatc atggtcatag ctgtttcctg
      301 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
      361 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
      421 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
      481 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
      541 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
      601 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
      661 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
      721 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
      781 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
      841 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
      901 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
      961 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     1021 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     1081 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     1141 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     1201 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     1261 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     1321 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     1381 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     1441 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     1501 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     1561 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     1621 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     1681 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     1741 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     1801 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     1861 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     1921 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     1981 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     2041 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     2101 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     2161 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     2221 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     2281 cgacacggaa atgttgaata ctcatactct tcctttttca attcagaaga actcgtcaag
     2341 aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa
     2401 gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca acgctatgtc
     2461 ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt
     2521 ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat cctcgccgtc
     2581 gggcatgcgc gccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgctcttc
     2641 gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc gctcgatgcg
     2701 atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat
     2761 tgcatcagcc atgatggata ctttctcggc aggagcaagg tgggatgaca ggagatcctg
     2821 ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac
     2881 agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcctgcag
     2941 ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga
     3001 cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa
     3061 tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg
     3121 aaacgatcct catcctgtct cttgatcaga tcttgatccc ctgcgccatc agatccttgg
     3181 cggcaagaaa gccatccagt ttactttgca gggcttccca accttaccag agggcgcccc
     3241 agctggcaat tccggttcgc ttgctgtcca taaaaccgcc cagtctagct atcgccatgt
     3301 aagcccactg caagctacct gctttctctt tgcgcttgcg ttttcccttg tccagatagc
     3361 ccagtagctg acattcatcc ggggtcagca ccgtttctgc ggactggctt tctacgtgtt
     3421 ccgcttcctt tagcagccct tgcgccctga attttgttaa aattcgcgtt aaatttttgt
     3481 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     3541 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     3601 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     3661 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     3721 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     3781 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     3841 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc cattcgccat
     3901 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
     3961 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
     4021 cacgacgttg taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg
     4081 gccctctaga tgcatgctcg agcggccgcc agtgtgatgg atttggaccc tcgtacagaa
     4141 gctaatacga ctcactatag ggaaataaga gagaaaagaa gagtaagaag aaatataaga
     4201 gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     4261 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     4321 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     4381 cccaccctcg tgaccaccct gacctatgga gtgcagtgct tcagccgcta ccccgaccac
     4441 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     4501 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     4561 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     4621 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     4681 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     4741 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     4801 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     4861 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     4921 aagtgaatcg ctgacttctg cggggcttgc ct
//
