LOCUS       sequence-386247-        4943 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient expression of mCherry; U6 with scaffold for sgRNA cloning 
            in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4943)
  AUTHORS   Spencley AL, Bar S, Swigut T, Flynn RA, Lee CH, Chen LF, Bassik MC, 
            Wysocka J
  TITLE     Co-transcriptional genome surveillance by HUSH is coupled to 
            termination machinery.
  JOURNAL   Mol Cell. 2023 May 18;83(10):1623-1639.e8. doi: 
            10.1016/j.molcel.2023.04.014. Epub 2023 May 9.
  PUBMED    37164018
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"; doi: 
            "10.1016/j.molcel.2023.04.014"; journalName: "Mol Cell"; date: 
            "2023-05-18- 18"; volume: "83"; issue: "10"; pages: "1623-1639.e8"
COMMENT     Sequence Label: pAS458-U6_CBA-mCherry
FEATURES             Location/Qualifiers
     source          1..4943
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(106..125)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      919..1507
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1408..1427
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        1569..1817
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1569..1589
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1740..1759
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1836..1911
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        2008..2293
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        2295..2571
                     /label=chicken beta-actin promoter
     intron          2572..2799
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      2811..2820
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2817..3524
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2962..2980)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(3552..3569)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3558..3765
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3774..3914
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3774..3903
                     /label=AAV2 ITR
     rep_origin      3989..4444
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4076..4095)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4286..4307
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4461..4480)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4580..4602
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4640..4658)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4726..4830
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4831..4899,4900..4943,1..748)
                     /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"
ORIGIN
        1 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
       61 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
      121 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
      181 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
      241 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
      301 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
      361 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
      421 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
      481 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
      541 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
      601 tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
      661 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
      721 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
      781 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
      841 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
      901 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
      961 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     1021 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     1081 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     1141 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     1201 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     1261 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     1321 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     1381 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     1441 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     1501 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     1561 tcacatgtga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
     1621 ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt
     1681 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
     1741 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
     1801 ggaaaggacg aaacaccggg tcttcgagaa gacctgtttt agagctagaa atagcaagtt
     1861 aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgtt
     1921 ttagagctag aaatagcaag ttaaaataag gctagtccgt ttttagcgcg tgcgccaatt
     1981 ctgcagacaa atggctctag aggtacccgt tacataactt acggtaaatg gcccgcctgg
     2041 ctgaccgccc aacgaccccc gcccattgac gtcaatagta acgccaatag ggactttcca
     2101 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
     2161 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcattg
     2221 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     2281 cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc
     2341 ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc
     2401 gggggggggg gggggcgcgc gccrggsggg gsggggsggg gsgrggggsg gggsggggsg
     2461 aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg
     2521 gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct
     2581 gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc
     2641 tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt
     2701 aattagctga gcaagaggta agggtttaag ggatggttgg ttggtggggt attaatgttt
     2761 aattacctgg agcacctgcc tgaaatcact ttttttcagg ttggaccggt gccaccatgg
     2821 tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc
     2881 acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
     2941 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttcg
     3001 cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg
     3061 ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga
     3121 tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg
     3181 agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc
     3241 agaagaaaac catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc
     3301 tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgctgagg
     3361 tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca
     3421 tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag tacgaacgcg
     3481 ccgagggccg ccactccacc ggcggcatgg acgagctgta caaggaattc taactagagc
     3541 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     3601 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     3661 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     3721 cagcaagggg gaggattggg aagagaatag caggcatgct ggggagcggc cgcaggaacc
     3781 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     3841 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     3901 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     3961 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     4021 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
     4081 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     4141 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     4201 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     4261 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     4321 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     4381 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     4441 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     4501 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     4561 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     4621 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     4681 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     4741 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     4801 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     4861 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     4921 gtaaaagatg ctgaagatca gtt
//
