LOCUS       sequence-441123-        4373 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  hU6-driven pre-gRNA plasmid for CasRx applications with puromycin 
            resistance. 5' processed DR followed by BbsI sites for guide cloning
            (Adapted from plasmid #10954).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4373)
  TITLE     LncRNA CISTR-ACT regulates cell size in human and mouse
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4373)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pXR004_puroR
FEATURES             Location/Qualifiers
     source          1..4373
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(240..828)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(320..339)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(999..1790,1791..1859))
                     /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     1622..1641
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1860..1964)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2032..2050
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2088..2110)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2210..2229
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2246..2701)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2383..2404)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2595..2614
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     misc_RNA        complement(2783..2818)
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        complement(2837..2872)
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     promoter        complement(2874..3122)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2932..2951)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3102..3122)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     polyA_signal    3171..3292
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3182..3201)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3236..3255
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3318..3917)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3401..3421)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     3898..3917
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(3942..4145)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3971..3991)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        join(4146..4373,1..152)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt cagccaggcg ggccatttac
       61 cgtaagttat gtaacgcgga actccatata tgggctatga actaatgacc ccgtaattga
      121 ttactattaa taactagtca ataatcaatg tcaacgcctc gagaattcag gccctctgct
      181 agctgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
      241 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
      301 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
      361 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
      421 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
      481 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
      541 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
      601 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
      661 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
      721 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
      781 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
      841 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
      901 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
      961 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     1021 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     1081 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcgg
     1141 cttccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     1201 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     1261 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     1321 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     1381 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     1441 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     1501 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     1561 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     1621 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     1681 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     1741 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     1801 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     1861 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     1921 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     1981 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     2041 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     2101 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     2161 cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag
     2221 cagattgtac tgagagtgca ccataaaatt gtaaacgtta atattttgtt aaaattcgcg
     2281 ttaaattttt gttaaatcag ctcatttttt aaccaataga ccgaaatcgg caaaatccct
     2341 tataaatcaa aagaatagcc cgagatagag ttgagtgttg ttccagtttg gaacaagagt
     2401 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     2461 ggcccactac gtgaaccatc acccaaatca agttttttgg ggtcgaggtg ccgtaaagca
     2521 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     2581 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctaaggcgct ggcaagtgta
     2641 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     2701 tactatggtt gctttgacgt atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     2761 accgcatcag aattcaaaaa aagtttcaaa ccccgaccag ttggtagggg tttacttgag
     2821 gtcttctcga agacccgttt caaaccccga ccagttggta ggggtttact tgcggtgttt
     2881 cgtcctttcc acaagatata taaagccaag aaatcgaaat actttcaagt tacggtaagc
     2941 atatgatagt ccattttaaa acataatttt aaaactgcaa actacccaag aaattattac
     3001 tttctacgtc acgtattttg tactaatatc tttgtgttta cagtcaaatt aattccaatt
     3061 atctctctaa cagccttgta tcgtatatgc aaatatgaag gaatcatggg aaataggccc
     3121 tcgctagctt cctgcccgac cttggtaccg gatcgaattt ccagacatga taagatacat
     3181 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     3241 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttagatctcg
     3301 acggtatcga tgaattctca ggcaccgggc ttgcgggtca tgcaccaggt gcgcggtcct
     3361 tcgggcacct cgacgtcggc ggtgacggtg aagccgagcc gctcgtagaa ggggaggttg
     3421 cggggcgcgg aggtctccag gaaggcgggc accccggcgc gctcggccgc ctccactccg
     3481 gggagcacga cggcgctgcc cagacccttg ccctggtggt cgggcgagac tccgacggtg
     3541 gccaggaacc acgcgggctc cttgggccgg tgcggcgcca ggaggccttc catctgttgc
     3601 tgcgcggcca gccgggaacc gctcaactcg gccatgcgcg ggccgatctc ggcgaacacc
     3661 gcccccgctt cgacgctctc cggcgtggtc cagaccgcca ccgcggcgcc gtcgtccgcg
     3721 acccacacct tgccgatgtc gagcccgacg cgcgtgagga agagttcttg cagctcggtg
     3781 acccgctcga tgtggcggtc cggatcgacg gtgtggcgcg tggcggggta gtcggcgaac
     3841 gcggcggcga gggtgcgtac ggccctgggg acgtcgtcgc gggtggcgag gcgcaccgtg
     3901 ggcttgtact cggtcatggt ggcaccggta gttagccaga gagctctgct tatatagacc
     3961 tcccaccgta cacgcctacc gcccatttgc gtcaatgggg cggagttgtt acgacatttt
     4021 ggaaagtccc gttgattttg gtgccaaaac aaactcccat tgacgtcaat ggggtggaga
     4081 cttggaaatc cccgtgagtc aaaccgctat ccacgcccat tgatgtactg ccaaaaccgc
     4141 atcaccatgg taatagcgat gactaatacg tagatgtact gccaagtagg aaagtcccat
     4201 aaggtcatgt actgggcata atgccaggcg ggccatttac cgtcattgac gtcaataggg
     4261 ggcgtacttg gcatatgata cacttgatgt actgccaagt gggcagttta ccgtaaatac
     4321 tccacccatt gacgtcaatg gaaagtccct attggcgtta ctatgggaac ata
//
