LOCUS       sequence-336490-        4942 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing a U6 promoter expressing a spyCas9 sgRNA 
            targeting B2M and CAG promoter expressing mTagBFP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4942)
  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 4942)
  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: pCAT009
FEATURES             Location/Qualifiers
     source          1..4942
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        453..738
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        740..1017
                     /label=chicken beta-actin promoter
     intron          1018..1245
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     primer_bind     complement(1983..2000)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1989..2196
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2205..2345
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2205..2334
                     /label=AAV2 ITR
     rep_origin      2420..2875
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2507..2526)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2717..2738
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2892..2911)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3011..3033
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3071..3089)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3157..3261
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3262..3330,3331..4122)
                     /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(3480..3499)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4293..4881
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4782..4801
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg gagtagcgcg agcacagcta gtttaagagc tatgctggaa acagcatagc
      301 aagtttaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
      361 ttgttttaga gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc
      421 caattctgca gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg
      481 cctggctgac cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact
      541 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      601 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      661 cattgtgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      721 gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc
      781 cccccctccc cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg
      841 ggggcggggg gggggggggg gcgcgcgccg gggggggggg ggsggggsgr ggggsggggs
      901 ggggsgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct
      961 tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga
     1021 gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc
     1081 cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg
     1141 ggctgtaatt agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta
     1201 atgtttaatt acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca
     1261 ccatgtctga actcatcaag gagaacatgc acatgaaact ctacatggag gggaccgtag
     1321 acaatcatca cttcaagtgt acaagtgaag gggaggggaa gccttacgaa gggactcaaa
     1381 ctatgagaat taaggttgtc gagggcggcc cccttccctt tgcgtttgac atcctcgcca
     1441 ccagtttctt gtatggttcc aagacgttta taaatcatac acagggaata cctgacttct
     1501 ttaaacaaag ttttccagag ggtttcacat gggaaagggt taccacttat gaggatggcg
     1561 gagtactcac cgctacccaa gatacatctc tgcaagatgg ttgcctgatt tacaatgtaa
     1621 aaatccgagg agttaacttt acatccaatg gtcctgttat gcagaaaaag acattggggt
     1681 gggaagcatt tacagaaacg ctctaccccg ccgacggggg cttggagggc cgcaatgaca
     1741 tggctctcaa attggtaggt ggtagtcatc ttatcgcgaa cgcgaaaact acctaccgat
     1801 ccaagaagcc cgctaaaaat ctcaaaatgc cggtctatta cgtggactac agactggaac
     1861 gcaaggaagc taacaacgag acatacgttg agcaacatga agtggccgtg gccagatatt
     1921 gcgatctgcc gagtaagctc gggcacaaat tgaactaaga attcctagag ctcgctgatc
     1981 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2041 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2101 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2161 ggaggattgg gaagagaata gcaggcatgc tggggagcgg ccgcaggaac ccctagtgat
     2221 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     2281 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
     2341 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     2401 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     2461 gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc
     2521 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     2581 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt
     2641 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     2701 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg
     2761 ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag
     2821 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg
     2881 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     2941 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     3001 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     3061 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     3121 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     3181 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     3241 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     3301 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     3361 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     3421 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     3481 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     3541 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     3601 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     3661 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     3721 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     3781 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     3841 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     3901 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     3961 ggagccggtg agcgtggaag ccgcggtatc attgcagcac tggggccaga tggtaagccc
     4021 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     4081 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     4141 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     4201 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     4261 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     4321 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     4381 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     4441 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     4501 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     4561 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     4621 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4681 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4741 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4801 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4861 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     4921 tgctggcctt ttgctcacat gt
//
