LOCUS       sequence-493703-        4972 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IVT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4972)
  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 4972)
  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"; doi: 
            "10.1038/s41586-025-09507-9"; journalName: "Nature"; date: 
            "2025-10"; volume: "646"; issue: "8086"; pages: "963-972"
COMMENT     Sequence Label: pPD0053_pIVTRup_EBFP2
FEATURES             Location/Qualifiers
     source          1..4972
                     /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(1278..1294)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1278..1294)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1291..1313)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1302..1318
                     /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(1326..1332,1333..1350,1351..1356))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1371..1392
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1509..1526)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1680..2268)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1760..1779)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2439..3230,3231..3299))
                     /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     3062..3081
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(3317..4111)
                     /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(3428..3447)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4038..4057
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      complement(4455..4883)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4565..4586)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4777..4796
                     /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 agctgttcac cggcgtggtg cccatcctgg tggagctgga cggcgacgtg
      301 aacggccaca agttcagcgt gaggggcgag ggcgagggcg acgccaccaa cggcaagctg
      361 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctggtgacc
      421 accctgagcc acggcgtgca gtgcttcgcc aggtaccccg accacatgaa gcagcacgac
      481 ttcttcaaga gcgccatgcc cgagggctac gtgcaggaga ggaccatctt cttcaaggac
      541 gacggcacct acaagaccag ggccgaggtg aagttcgagg gcgacaccct ggtgaacagg
      601 atcgagctga agggcgtgga cttcaaggag gacggcaaca tcctgggcca caagctggag
      661 tacaacttca acagccacaa catctacatc atggccgtga agcagaagaa cggcatcaag
      721 gtgaacttca agatcaggca caacgtggag gacggcagcg tgcagctggc cgaccactac
      781 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacagcca ctacctgagc
      841 acccagagcg tgctgagcaa ggaccccaac gagaagaggg accacatggt gctgctggag
      901 ttcaggaccg ccgccggcat caccctgggc atggacgagc tgtacaagtg aatcgctgcc
      961 ttctgcgggg cttgccttct ggccatgccc ttcttctctc ccttgcacct gtacctcttg
     1021 gtctttgaat aaagcctgag taggaagtga gggtctagaa aaaaaaaaaa aaaaaaaaaa
     1081 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     1141 aaaaaaaaaa aaaaaaaaaa aaacgatcaa taaccgacac tcctgcgtcg cctacgcgct
     1201 acgatctgca gaattccagc acactggcgg ccgttactag tggatccgag ctcggtacca
     1261 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     1321 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     1381 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     1441 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     1501 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     1561 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     1621 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     1681 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     1741 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     1801 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     1861 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     1921 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     1981 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     2041 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     2101 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     2161 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     2221 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     2281 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     2341 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     2401 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     2461 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     2521 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     2581 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     2641 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     2701 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     2761 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     2821 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     2881 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     2941 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     3001 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     3061 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     3121 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     3181 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     3241 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     3301 ctcttccttt ttcaattcag aagaactcgt caagaaggcg atagaaggcg atgcgctgcg
     3361 aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg ccgccaagct
     3421 cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc acacccagcc
     3481 ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc ggcaagcagg
     3541 catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg agcctggcga
     3601 acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac
     3661 cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc
     3721 aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct
     3781 cggcaggagc aaggtgggat gacaggagat cctgccccgg cacttcgccc aatagcagcc
     3841 agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg
     3901 ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg
     3961 tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc
     4021 agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag
     4081 aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct gtctcttgat
     4141 cagatcttga tcccctgcgc catcagatcc ttggcggcaa gaaagccatc cagtttactt
     4201 tgcagggctt cccaacctta ccagagggcg ccccagctgg caattccggt tcgcttgctg
     4261 tccataaaac cgcccagtct agctatcgcc atgtaagccc actgcaagct acctgctttc
     4321 tctttgcgct tgcgttttcc cttgtccaga tagcccagta gctgacattc atccggggtc
     4381 agcaccgttt ctgcggactg gctttctacg tgttccgctt cctttagcag cccttgcgcc
     4441 ctgaattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     4501 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     4561 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     4621 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     4681 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     4741 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     4801 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     4861 taatgcgccg ctacagggcg cgtccattcg ccattcaggc tgcgcaactg ttgggaaggg
     4921 cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tg
//
