LOCUS       sequence-332770-        4943 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 4943)
  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 4943)
  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..4943
                     /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..1246
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     primer_bind     complement(1984..2001)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1990..2197
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2206..2346
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2206..2335
                     /label=AAV2 ITR
     rep_origin      2421..2876
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2508..2527)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2718..2739
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2893..2912)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3012..3034
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3072..3090)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3158..3262
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3263..3331,3332..4123)
                     /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(3481..3500)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4294..4882
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4783..4802
                     /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 gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc
      901 ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct
      961 tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga
     1021 gtcgctgcga cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc
     1081 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
     1141 gggctgtaat tagctgagca agaggtaagg gtttaaggga tggttggttg gtggggtatt
     1201 aatgtttaat tacctggagc acctgcctga aatcactttt tttcaggttg gaccggtgcc
     1261 accatgtctg aactcatcaa ggagaacatg cacatgaaac tctacatgga ggggaccgta
     1321 gacaatcatc acttcaagtg tacaagtgaa ggggagggga agccttacga agggactcaa
     1381 actatgagaa ttaaggttgt cgagggcggc ccccttccct ttgcgtttga catcctcgcc
     1441 accagtttct tgtatggttc caagacgttt ataaatcata cacagggaat acctgacttc
     1501 tttaaacaaa gttttccaga gggtttcaca tgggaaaggg ttaccactta tgaggatggc
     1561 ggagtactca ccgctaccca agatacatct ctgcaagatg gttgcctgat ttacaatgta
     1621 aaaatccgag gagttaactt tacatccaat ggtcctgtta tgcagaaaaa gacattgggg
     1681 tgggaagcat ttacagaaac gctctacccc gccgacgggg gcttggaggg ccgcaatgac
     1741 atggctctca aattggtagg tggtagtcat cttatcgcga acgcgaaaac tacctaccga
     1801 tccaagaagc ccgctaaaaa tctcaaaatg ccggtctatt acgtggacta cagactggaa
     1861 cgcaaggaag ctaacaacga gacatacgtt gagcaacatg aagtggccgt ggccagatat
     1921 tgcgatctgc cgagtaagct cgggcacaaa ttgaactaag aattcctaga gctcgctgat
     1981 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     2041 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     2101 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     2161 gggaggattg ggaagagaat agcaggcatg ctggggagcg gccgcaggaa cccctagtga
     2221 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
     2281 tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc
     2341 tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     2401 tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     2461 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     2521 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     2581 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg
     2641 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     2701 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     2761 gggctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     2821 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg
     2881 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     2941 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     3001 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     3061 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     3121 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     3181 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     3241 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     3301 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     3361 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     3421 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     3481 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     3541 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     3601 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     3661 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     3721 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     3781 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     3841 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     3901 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     3961 tggagccggt gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc
     4021 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     4081 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     4141 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     4201 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     4261 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     4321 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     4381 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     4441 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     4501 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     4561 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     4621 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     4681 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     4741 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     4801 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     4861 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     4921 ttgctggcct tttgctcaca tgt
//
