LOCUS       sequence-270571-        4838 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a guide RNA targeting HEK3 site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4838)
  AUTHORS   McGrath E, Shin H, Zhang L, Phue JN, Wu WW, Shen RF, Jang YY, 
            Revollo J, Ye Z
  TITLE     Targeting specificity of APOBEC-based cytosine base editor in human 
            iPSCs determined by whole genome sequencing.
  JOURNAL   Nat Commun. 2019 Nov 25;10(1):5353. doi: 10.1038/s41467-019-13342-8.
  PUBMED    31767844
REFERENCE   2  (bases 1 to 4838)
  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: "Nat 
            Commun."; date: "2019-11-25"; volume: "10(1):5353. doi"; pages: " 
            10.1038/s41467-019-13342-8"
COMMENT     Sequence Label: gRNA-HEK3-Puro
FEATURES             Location/Qualifiers
     source          1..4838
                     /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"
     misc_RNA        270..345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        442..727
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1007..1234
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1252..1851
                     /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(1252..1271)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1748..1768
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1879..1896)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1885..2092
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2101..2241
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2101..2230
                     /label=AAV2 ITR
     rep_origin      2316..2771
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2403..2422)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2613..2634
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2788..2807)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2907..2929
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2967..2985)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3053..3157
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3158..3226,3227..4018)
                     /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(3376..3395)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4189..4777
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4678..4697
                     /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 gcccagactg agcacgtgag ttttagagct agaaatagca agttaaaata
      301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag
      361 ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag
      421 acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc
      481 gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg
      541 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      601 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca
      661 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
      721 taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
      781 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
      841 gggggggggg cgcgcgccrg gsgggggggg gggggggggg gggggggggg ggggggggsg
      901 gggrggkgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
      961 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg
     1021 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
     1081 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta
     1141 gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta
     1201 cctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtgccac catgaccgag
     1261 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     1321 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     1381 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     1441 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     1501 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     1561 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     1621 ttcctggcca ccgtcggagt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     1681 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     1741 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     1801 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg agaattctaa
     1861 ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     1921 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     1981 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     2041 ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg gagcggccgc
     2101 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     2161 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     2221 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
     2281 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     2341 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc
     2401 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2461 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2521 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2581 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2641 actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta
     2701 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     2761 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     2821 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     2881 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     2941 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     3001 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     3061 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     3121 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     3181 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     3241 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     3301 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     3361 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     3421 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     3481 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     3541 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     3601 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     3661 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     3721 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     3781 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     3841 tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg
     3901 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     3961 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     4021 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     4081 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     4141 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4201 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4261 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4321 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4381 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4441 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4501 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4561 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4621 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4681 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4741 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4801 acggttcctg gccttttgct ggccttttgc tcacatgt
//
