LOCUS       sequence-450764-        6527 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV-Intron-BRL (env protein). Expresses BRL (BaEVRLess) env for VLP 
            production..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6527)
  AUTHORS   Ramadoss GN, Namaganda SJ, Kumar MM, Hamilton JR, Sharma R, Chow KG,
            Workley LA, Macklin BL, Sun M, Ha AS, Liu JC, Fellmann C, Watry HL, 
            Dierks PH, Bose RS, Jin J, Perez BS, Sandoval Espinoza CR, Matia MP,
            Lu SH, Judge LM, Shy BR, Nussenzweig A, Adamson B, Murthy N, Doudna 
            JA, Kampmann M, Conklin BR
  TITLE     Characterizing and controlling CRISPR repair outcomes in nondividing
            human cells.
  JOURNAL   Nat Commun. 2025 Nov 17;16(1):9883. doi: 10.1038/s41467-025-66058-3.
  PUBMED    41249169
REFERENCE   2  (bases 1 to 6527)
  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"; journalName: "Nat 
            Commun."; date: "2025-11-17"; volume: "16(1):9883. doi"; pages: " 
            10.1038/s41467-025-66058-3"
COMMENT     Sequence Label: pCF159_BRL
FEATURES             Location/Qualifiers
     source          1..6527
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        97..476
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        477..680
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     631..651
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     677..701
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          792..1364
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     primer_bind     complement(3232..3251)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3297..3352
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3351..3370)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3710..3726)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3710..3726)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3723..3745)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3734..3750
                     /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(3758..3764,3765..3782,3783..3788))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3803..3824
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3941..3958)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4112..4700)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4192..4211)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4871..5662,5663..5731))
                     /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     5494..5513
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5732..5836)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5862..6317)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5999..6020)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6211..6230
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6444..6466
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6458..6475
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6459..6475
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6485..6504
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6485..6503
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gcggccgctc tagagagctt ggcccattgc atacgttgta tccatatcat aatatgtaca
       61 tttatattgg ctcatgtcca acattaccgc catgttgaca ttgattattg actagttatt
      121 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
      181 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      241 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      301 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
      361 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
      421 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
      481 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
      541 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
      601 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
      661 aggtctatat aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac
      721 gctgttttga cctccataga agacaccggg accgatccag cctccggtcg accgatcctg
      781 agaacttcag ggtgagtttg gggacccttg attgttcttt ctttttcgct attgtaaaat
      841 tcatgttata tggagggggc aaagttttca gggtgttgtt tagaatggga agatgtccct
      901 tgtatcacca tggaccctca tgataatttt gtttctttca ctttctactc tgttgacaac
      961 cattgtctcc tcttattttc ttttcatttt ctgtaacttt ttcgttaaac tttagcttgc
     1021 atttgtaacg aatttttaaa ttcacttttg tttatttgtc agattgtaag tactttctct
     1081 aatcactttt ttttcaaggc aatcagggta tattatattg tacttcagca cagttttaga
     1141 gaacaattgt tataattaaa tgataaggta gaatatttct gcatataaat tctggctggc
     1201 gtggaaatat tcttattggt agaaacaact acaccctggt catcatcctg cctttctctt
     1261 tatggttaca atgatataca ctgtttgaga tgaggataaa atactctgag tccaaaccgg
     1321 gcccctctgc taaccatgtt catgccttct tctctttcct acagctcctg ggcaacgtgc
     1381 tggttgttgt gctgtctcat cattttggca aagctcgagc tgtatttgtg ccaacatggg
     1441 attcacaaca aagataatct tcttatacaa cctagtactg gtctacgcgg ggtttgacga
     1501 ccctcgcaaa gccatagaac tagtacaaaa gcgatatggc cgaccatgcg attgcagcgg
     1561 aggacaagtg tccgagcccc cgtcagacag ggtcagtcaa gtgacttgct caggcaagac
     1621 agcttactta atgcccgacc aaagatggaa atgtaagtca attccaaaag acacctcccc
     1681 aagcgggcca ctccaagagt gcccctgtaa ttcttaccag tcctcagtac acagttcttg
     1741 ttatacctca taccaacaat gcagatcagg caataagaca tattatacgg ctactctgct
     1801 aaaaacacaa actgggggca ccagtgatgt acaagtatta ggatccacca acaaacttat
     1861 acaatctccc tgtaatggca taaaagggca gtctatttgc tggagcacta cagctcctat
     1921 ccacgtctct gatggaggag gtccattaga caccacaaga attaaaagtg ttcagagaaa
     1981 actggaagaa attcataaag ccctatatcc tgaacttcag tatcaccctt tggccatacc
     2041 taaggttaga gataacctca tggtcgatgc ccagacttta aacattctca atgccactta
     2101 caacttactc ctaatgtcca acacgagcct agtggacgac tgttggcttt gtttaaaatt
     2161 aggtccccct actcccctcg caatacctaa cttcctatta tcctacgtga ctcgctcctc
     2221 ggataatatc tcttgtttaa taattccccc ccttctagtt caaccgatgc agttttccaa
     2281 ttcatcttgc ctcttttccc cctcctacaa cagtacagaa gaaatagatc taggccatgt
     2341 tgccttcagc aactgtacct ccataaccaa tgtcaccggt cccatatgcg ctgtaaatgg
     2401 ttcggtcttt ctctgtggca ataacatggc atacacttat ctacccacga actggacggg
     2461 gctttgcgtc ctagcaactc tcctccccga cattgacatc attcccggag atgaaccggt
     2521 ccccatccct gctattgatc attttatata tagacctaaa cgggccatac agtttattcc
     2581 tttactagca gggctaggga tcaccgcagc cttcacaaca ggagctacag gcctaggtgt
     2641 ctctgtgacc caatatacaa aattatctaa tcagctaatt tctgatgtac aaatcttatc
     2701 tagcaccata caagatctgc aagatcaagt agactcatta gccgaagtgg ttctccagaa
     2761 cagaaggggg ctagatctac ttacagcaga acaaggagga atctgtttag ccctgcaaga
     2821 aaaatgctgc ttttatgtta acaagtcagg gattgtgaga gacaaaataa aaaccttaca
     2881 agaagaacta gaaagacgta gaaaagatct agcttccaac ccactttgga ctgggcttca
     2941 agggctcctc ccttacctcc tgccctttct tggccctcta cttaccctcc tgctcttact
     3001 caccattggg ccgtgcattt ttaaccgtct aaccgctttt attaatgata agttaaacat
     3061 aatacacgct atgtagttaa cccaacagta tcaggtgctc agaaccgatg aagaagctca
     3121 agattgagcc tctaagacac aagaaaagga attcactcct caggtgcagg ctgcctatca
     3181 gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa taccactgag atctttttcc
     3241 ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata
     3301 aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag
     3361 gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc
     3421 aacatatgcc catatgctgg ctgccatgaa caaaggttgg ctataaagag gtcatcagta
     3481 tatgaaacag ccccctgctg tccattcctt attccataga aaagccttga cttgaggtta
     3541 gatttttttt atattttgtt ttgtgttatt tttttcttta acatccctaa aattttcctt
     3601 acatgtttta ctagccagat ttttcctcct ctcctgacta ctcccagtca tagctgtccc
     3661 tcttctctta tggagatccc tcgacggatc ggccgcaatt cgtaatcatg tcatagctgt
     3721 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     3781 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     3841 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     3901 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     3961 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4021 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     4081 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4141 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     4201 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4261 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4321 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4381 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4441 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4501 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     4561 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4621 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     4681 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4741 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     4801 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     4861 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     4921 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     4981 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     5041 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5101 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5161 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     5221 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     5281 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     5341 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     5401 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     5461 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     5521 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     5581 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     5641 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     5701 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     5761 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     5821 aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat
     5881 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     5941 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     6001 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     6061 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     6121 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     6181 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     6241 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     6301 gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc
     6361 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
     6421 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg
     6481 cgcgtaatac gactcactat agggcgaatt ggagctccac cgcggtg
//
