LOCUS       sequence-440390-        4331 bp    DNA     circular SYN 14-APR-2025
DEFINITION  hU6-driven sgRNA plasmid for CasRx applications with puromycin 
            resistance. 5' processed DR followed by BbsI sites for guide cloning
            (Adapted from plasmid #109053).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4331)
  TITLE     LncRNA CISTR-ACT regulates cell size in human and mouse
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4331)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pXR003_puroR
FEATURES             Location/Qualifiers
     source          1..4331
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        17..396
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        397..600
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     551..571
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             625..1224
                     /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(625..644)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1121..1141
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1250..1371
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1287..1306)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1341..1360
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1420..1668
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1420..1440
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1591..1610
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1670..1699
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     rep_origin      1799..2254
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1886..1905)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2096..2117
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2271..2290)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2390..2412
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2450..2468)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2536..2640
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2641..2709,2710..3501)
                     /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(2859..2878)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3672..4260
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4161..4180
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaattctcga ggcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg
       61 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
      121 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
      181 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      241 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      301 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
      361 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
      421 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
      481 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
      541 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
      601 ctctggctaa ctaccggtgc caccatgacc gagtacaagc ccacggtgcg cctcgccacc
      661 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
      721 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
      781 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
      841 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
      901 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
      961 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg agtctcgccc
     1021 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     1081 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     1141 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     1201 atgacccgca agcccggtgc ctgagaattc atcgataccg tcgagatcta acttgtttat
     1261 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     1321 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     1381 gaaattcgat ccggtaccaa ggtcgggcag gaagctagcg agggcctatt tcccatgatt
     1441 ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat taatttgact
     1501 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
     1561 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
     1621 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccga acccctacca
     1681 actggtcggg gtttgaaacg ggtcttcgag aagacctttt ttttgaattc tgatgcggta
     1741 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac
     1801 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     1861 acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     1921 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     1981 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca
     2041 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     2101 ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa
     2161 gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     2221 gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc
     2281 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     2341 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     2401 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
     2461 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     2521 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     2581 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     2641 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     2701 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     2761 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     2821 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     2881 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     2941 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     3001 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     3061 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     3121 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     3181 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     3241 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     3301 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtggaagc
     3361 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     3421 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     3481 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     3541 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     3601 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     3661 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     3721 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     3781 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     3841 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     3901 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     3961 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     4021 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     4081 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4141 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4201 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     4261 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcagcta
     4321 gcagagggcc t
//
