LOCUS       sequence-354439-        4887 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing a U6 promoter expressing a spyCas9 tdTom sgRNA298
            and CAG promoter expressing mTagBFP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4887)
  AUTHORS   Hamilton JR, Tsuchida CA, Nguyen DN, Shy BR, McGarrigle ER, Sandoval
            Espinoza CR, Carr D, Blaeschke F, Marson A, Doudna JA
  TITLE     Targeted delivery of CRISPR-Cas9 and transgenes enables complex 
            immune cell engineering.
  JOURNAL   Cell Rep. 2021 Jun 1;35(9):109207. doi: 
            10.1016/j.celrep.2021.109207.
  PUBMED    34077734
REFERENCE   2  (bases 1 to 4887)
  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: "Cell Rep.";
            date: "2021-06-1"; volume: "35(9):109207. doi"; pages: " 
            10.1016/j.celrep.2021.109207"
COMMENT     Sequence Label: pJRH056
FEATURES             Location/Qualifiers
     source          1..4887
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          107..334
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     primer_bind     complement(1072..1089)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1078..1285
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   1294..1434
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1294..1423
                     /label=AAV2 ITR
     rep_origin      1509..1964
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1596..1615)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1806..1827
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1981..2000)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2100..2122
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2160..2178)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2246..2350
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2351..2419,2420..3211)
                     /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     complement(2569..2588)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3382..3970
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3871..3890
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4032..4280
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4032..4052
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4203..4222
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        4484..4769
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
       61 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg
      121 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
      181 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta
      241 gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta
      301 cctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtgccac catgtctgaa
      361 ctcatcaagg agaacatgca catgaaactc tacatggagg ggaccgtaga caatcatcac
      421 ttcaagtgta caagtgaagg ggaggggaag ccttacgaag ggactcaaac tatgagaatt
      481 aaggttgtcg agggcggccc ccttcccttt gcgtttgaca tcctcgccac cagtttcttg
      541 tatggttcca agacgtttat aaatcataca cagggaatac ctgacttctt taaacaaagt
      601 tttccagagg gtttcacatg ggaaagggtt accacttatg aggatggcgg agtactcacc
      661 gctacccaag atacatctct gcaagatggt tgcctgattt acaatgtaaa aatccgagga
      721 gttaacttta catccaatgg tcctgttatg cagaaaaaga cattggggtg ggaagcattt
      781 acagaaacgc tctaccccgc cgacgggggc ttggagggcc gcaatgacat ggctctcaaa
      841 ttggtaggtg gtagtcatct tatcgcgaac gcgaaaacta cctaccgatc caagaagccc
      901 gctaaaaatc tcaaaatgcc ggtctattac gtggactaca gactggaacg caaggaagct
      961 aacaacgaga catacgttga gcaacatgaa gtggccgtgg ccagatattg cgatctgccg
     1021 agtaagctcg ggcacaaatt gaactaagaa ttcctagagc tcgctgatca gcctcgactg
     1081 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     1141 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     1201 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     1261 aagagaatag caggcatgct ggggagcggc cgcaggaacc cctagtgatg gagttggcca
     1321 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     1381 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc
     1441 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca
     1501 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     1561 cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt
     1621 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     1681 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg
     1741 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     1801 taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt
     1861 tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca
     1921 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag
     1981 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     2041 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     2101 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     2161 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     2221 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2281 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     2341 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     2401 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     2461 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     2521 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     2581 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     2641 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     2701 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     2761 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     2821 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     2881 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     2941 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3001 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3061 gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3121 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     3181 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     3241 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     3301 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3361 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3421 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3481 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     3541 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3601 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3661 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3721 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     3781 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     3841 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3901 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     3961 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4021 tgctcacatg tgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
     4081 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
     4141 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     4201 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
     4261 tgtggaaagg acgaaacacc gaagtaaaac ctctacaaat ggtttaagag ctatgctgga
     4321 aacagcatag caagtttaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
     4381 tcggtgcttt tttgttttag agctagaaat agcaagttaa aataaggcta gtccgttttt
     4441 agcgcgtgcg ccaattctgc agacaaatgg ctctagaggt acccgttaca taacttacgg
     4501 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca atagtaacgc
     4561 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     4621 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
     4681 ggcccgcctg gcattgtgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     4741 tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc
     4801 tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt
     4861 gtgcagcgat gggggcgggg ggggggg
//
