LOCUS       sequence-345453-        6705 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing paired pegRNAs from human U6 and H1 promoters to make 
            252-bp deletion on HPRT1 gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6705)
  AUTHORS   Choi J, Chen W, Suiter CC, Lee C, Chardon FM, Yang W, Leith A, Daza 
            RM, Martin B, Shendure J
  TITLE     Precise genomic deletions using paired prime editing.
  JOURNAL   Nat Biotechnol. 2021 Oct 14. pii: 10.1038/s41587-021-01025-z. doi: 
            10.1038/s41587-021-01025-z.
  PUBMED    34650269
REFERENCE   2  (bases 1 to 6705)
  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 
            Biotechnol. 2021 Oct 14. pii: 10.1038/s41587-021-01025-z. doi: 
            10.1038/s41587-021-01025-z."
COMMENT     Sequence Label: PB-HD252
FEATURES             Location/Qualifiers
     source          1..6705
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(166..754)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(246..265)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(925..1716,1717..1785))
                     /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     1548..1567
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1786..1890)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1916..2371)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2053..2074)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2265..2284
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2513..2529
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   2659..2721
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        3146..3394
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3146..3166
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3317..3336
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3416..3491
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     3559..3575
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3717..3736
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     misc_RNA        3826..3901
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4025..4236
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             4249..4517
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             4543..4938
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(4998..5018)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    5060..5181
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5097..5116)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5151..5170
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5944..5978
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6469..6485)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6469..6485)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6482..6504)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6493..6509
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(6517..6523,6524..6541,6542..6547))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6562..6583
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(join(6700..6705,1..12))
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
       61 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
      121 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
      181 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
      241 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
      301 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
      361 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
      421 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
      481 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
      541 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
      601 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
      661 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
      721 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
      781 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
      841 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
      901 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
      961 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     1021 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     1081 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     1141 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     1201 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     1261 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     1321 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     1381 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     1441 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     1501 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     1561 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     1621 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     1681 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     1741 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     1801 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     1861 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt
     1921 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     1981 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     2041 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     2101 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     2161 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     2221 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     2281 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     2341 gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt
     2401 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
     2461 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
     2521 acggccagtg agcgcgcctc gttcattcac gtttttgaac ccgtggagga cgggcagact
     2581 cgcggtgcaa atgtgtttta cagcgtgatg gagcagatga agatgctcga cacgctgcag
     2641 aacacgcagc tagattaacc ctagaaagat aatcatattg tgacgtacgt taaagataat
     2701 catgtgtaaa attgacgcat gtgttttatc ggtctgtata tcgaggttta tttattaatt
     2761 tgaatagata ttaagtttta ttatatttac acttacatac taataataaa ttcaacaaac
     2821 aatttattta tgtttattta tttattaaaa aaaacaaaaa ctcaaaattt cttctataaa
     2881 gtaacaaaac ttttatgagg gacagccccc ccccaaagcc cccagggatg taattacgtc
     2941 cctcccccgc tagggggcag cagcgagccg cccggggctc cgctccggtc cggcgctccc
     3001 cccgcatccc cgagccggca gcgtgcgggg acagcccggg cacggggaag gtggcacggg
     3061 atcgctttcc tctgaacgct tctcgctgct ctttgagcct gcagacacct ggggggatac
     3121 ggggaaaagg cctccacggc caagagaggg cctatttccc atgattcctt catatttgca
     3181 tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat
     3241 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
     3301 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
     3361 ggctttatat atcttgtgga aaggacgaaa caccggcctg caaactggta ggcgcgtttt
     3421 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac
     3481 cgagtcggtg cagggccggc aggccgagct gctcaccacg acgcctacca gtttgctttt
     3541 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgaacgctga cgtcatcaac
     3601 ccgctccaag gaatcgcggg cccagtgtca ctaggcggga acacccagcg cgcgtgcgcc
     3661 ctggcaggaa gatggctgtg agggacaggg gagtggcgcc ctgcaatatt tgcatgtcgc
     3721 tatgtgttct gggaaatcac cataaacgtg aaatgtcttt ggatttggga atcttataag
     3781 ttctgtatga ggaccacaga tccccagctg ctcaccacga cgccagtttt agagctagaa
     3841 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
     3901 cacggctacc tagtgagcct gcaaactggt aggcgtcgtg gtgagctttt ttaagcttgg
     3961 cgtaactaga tcttgagaca aacgcggccg cgaaggatct gcgatcgctc cggtgcccgt
     4021 cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat
     4081 tgaacgggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg
     4141 ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac
     4201 gttctttttc gcaacgggtt tgccgccaga acacagctga agcttcgagg ggctcgcatc
     4261 tctccttcac gcgcccgccg ccctacctga ggccgccatc cacgccggtt gagtcgcgtt
     4321 ctgccgcctc ccgcctgtgg tgcctcctga actgcgtccg ccgtctaggt aagtttaaag
     4381 ctcaggtcga gaccgggcct ttgtccggcg ctcccttgga gcctacctag actcagccgg
     4441 ctctccacgc tttgcctgac cctgcttgct caactctacg tctttgtttc gttttctgtt
     4501 ctgcgccgtt acagatccaa gctgtgaccg gcgcctaccg tcatggccaa gcctttgtct
     4561 caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct
     4621 gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc
     4681 aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct
     4741 gcggcagctg gcaacctgac ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg
     4801 agcccctgcg gacggtgccg acaggtgctt ctcgatctgc atcctgggat caaagccata
     4861 gtgaaggaca gtgatggaca gccgacggca gttgggattc gtgaattgct gccctctggt
     4921 tatgtgtggg agggctaaac gcgtaatcaa cctctggatt acaaaatttg tgaaagattg
     4981 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     5041 ttgtatcatg cgttaactaa acttgtttat tgcagcttat aatggttaca aataaagcaa
     5101 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     5161 caaactcatc aatgtatctt atcatgtctg gaattgactc aaatgatgtc aattagtcta
     5221 tcagaagctc atctggtctc ccttccgggg gacaagacat ccctgtttaa tatttaaaca
     5281 gcagtgttcc caaactgggt tcttatatcc cttgctctgg tcaaccaggt tgcagggttt
     5341 cctgtcctca caggaacgaa gtccctaaag aaacagtggc agccaggttt agccccggaa
     5401 ttgactggat tcctttttta gggcccattg gtatggcttt ttccccgtat ccccccaggt
     5461 gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg tgccaccttc
     5521 cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga gcgccggacc
     5581 ggagcggagc cccgggcggc tcgctgctgc cccctagcgg gggagggacg taattacatc
     5641 cctgggggct ttgggggggg gctgtccctg atatctataa caagaaaata tatatataat
     5701 aagttatcac gtaagtagaa catgaaataa caatataatt atcgtatgag ttaaatctta
     5761 aaagtcacgt aaaagataat catgcgtcat tttgactcac gcggtcgtta tagttcaaaa
     5821 tcagtgacac ttaccgcatt gacaagcacg cctcacggga gctccaagcg gcgactgaga
     5881 tgtcctaaat gcacagcgac ggattcgcgc tatttagaaa gagagagcaa tatttcaaga
     5941 atgcatgcgt caattttacg cagactatct ttctagggtt aatctagctg catcaggatc
     6001 atatcgtcgg gtcttttttc cggctcagtc atcgcccaag ctggcgctat ctgggcatcg
     6061 gggaggaaga agcccgtgcc ttttcccgcg aggttgaagc ggcatggaaa gagtttgccg
     6121 aggatgactg ctgctgcatt gacgttgagc gaaaacgcac gtttaccatg atgattcggg
     6181 aaggtgtggc catgcacgcc tttaacggtg aactgttcgt tcaggccacc tgggatacca
     6241 gttcgtcgcg gcttttccgg acacagttcc ggatggtcag cccgaagcgc atcagcaacc
     6301 cgaacaatac cggcgacagc cggaactgcc gtgccggtgt gcagattaat gacagcggtg
     6361 cggcgctggg atattacgtc agcgaggacg ggtatcctgg ctggatgccg cagaaatgga
     6421 catggatacc ccgtgagtta cccggcgggc gcgcttggcg taatcatggt catagctgtt
     6481 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     6541 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     6601 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     6661 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgct
//
