LOCUS       sequence-345455-        6706 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing paired pegRNAs from human U6 and H1 promoters to make 
            118-bp deletion on HPRT1 gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6706)
  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 6706)
  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-HD118
FEATURES             Location/Qualifiers
     source          1..6706
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(64..81)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(235..823)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(315..334)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(994..1785,1786..1854))
                     /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     1617..1636
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1855..1959)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1985..2440)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2122..2143)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2334..2353
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2582..2598
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   2728..2790
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        3215..3463
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3215..3235
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3386..3405
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3485..3560
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     3628..3644
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3786..3805
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     misc_RNA        3895..3970
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4095..4306
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             4319..4587
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             4613..5008
                     /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(5068..5088)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    5130..5251
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5167..5186)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5221..5240
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   6014..6048
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6539..6555)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6539..6555)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6552..6574)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6563..6579
                     /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(6587..6593,6594..6611,6612..6617))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6632..6653
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
       61 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
      121 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
      181 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
      241 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
      301 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
      361 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
      421 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
      481 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
      541 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
      601 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
      661 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
      721 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
      781 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
      841 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
      901 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
      961 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     1021 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     1081 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     1141 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     1201 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     1261 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     1321 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     1381 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     1441 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     1501 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     1561 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     1621 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     1681 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     1741 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     1801 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     1861 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     1921 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     1981 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     2041 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     2101 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     2161 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     2221 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     2281 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     2341 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     2401 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg
     2461 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa
     2521 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt
     2581 tgtaaaacga cggccagtga gcgcgcctcg ttcattcacg tttttgaacc cgtggaggac
     2641 gggcagactc gcggtgcaaa tgtgttttac agcgtgatgg agcagatgaa gatgctcgac
     2701 acgctgcaga acacgcagct agattaaccc tagaaagata atcatattgt gacgtacgtt
     2761 aaagataatc atgtgtaaaa ttgacgcatg tgttttatcg gtctgtatat cgaggtttat
     2821 ttattaattt gaatagatat taagttttat tatatttaca cttacatact aataataaat
     2881 tcaacaaaca atttatttat gtttatttat ttattaaaaa aaacaaaaac tcaaaatttc
     2941 ttctataaag taacaaaact tttatgaggg acagcccccc cccaaagccc ccagggatgt
     3001 aattacgtcc ctcccccgct agggggcagc agcgagccgc ccggggctcc gctccggtcc
     3061 ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg
     3121 tggcacggga tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg
     3181 gggggatacg gggaaaaggc ctccacggcc aagagagggc ctatttccca tgattccttc
     3241 atatttgcat atacgataca aggctgttag agagataatt agaattaatt tgactgtaaa
     3301 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc
     3361 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt
     3421 cgatttcttg gctttatata tcttgtggaa aggacgaaac accgaacctc tcggctttcc
     3481 cgcggtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
     3541 aagtggcacc gagtcggtgc agggccggca ggccgagctg ctcaccacga cggggaaagc
     3601 cgagattttt ttcccagtca cgacgttgta aaacgacggc cagtgaattc gaacgctgac
     3661 gtcatcaacc cgctccaagg aatcgcgggc ccagtgtcac taggcgggaa cacccagcgc
     3721 gcgtgcgccc tggcaggaag atggctgtga gggacagggg agtggcgccc tgcaatattt
     3781 gcatgtcgct atgtgttctg ggaaatcacc ataaacgtga aatgtctttg gatttgggaa
     3841 tcttataagt tctgtatgag gaccacagat ccccagctgc tcaccacgac gccagtttta
     3901 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
     3961 gagtcggtgc acgagccctc aggcgaacct ctcggctttc cccgtcgtgg tgagcttttt
     4021 ttaagcttgg cgtaactaga tcttgagaca aacgcggccg cgaaggatct gcgatcgctc
     4081 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
     4141 ggtcggcaat tgaacgggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
     4201 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
     4261 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacagctga agcttcgagg
     4321 ggctcgcatc tctccttcac gcgcccgccg ccctacctga ggccgccatc cacgccggtt
     4381 gagtcgcgtt ctgccgcctc ccgcctgtgg tgcctcctga actgcgtccg ccgtctaggt
     4441 aagtttaaag ctcaggtcga gaccgggcct ttgtccggcg ctcccttgga gcctacctag
     4501 actcagccgg ctctccacgc tttgcctgac cctgcttgct caactctacg tctttgtttc
     4561 gttttctgtt ctgcgccgtt acagatccaa gctgtgaccg gcgcctaccg tcatggccaa
     4621 gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat
     4681 ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt
     4741 cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg
     4801 cactgctgct gcggcagctg gcaacctgac ttgtatcgtc gcgatcggaa atgagaacag
     4861 gggcatcttg agcccctgcg gacggtgccg acaggtgctt ctcgatctgc atcctgggat
     4921 caaagccata gtgaaggaca gtgatggaca gccgacggca gttgggattc gtgaattgct
     4981 gccctctggt tatgtgtggg agggctaaac gcgtaatcaa cctctggatt acaaaatttg
     5041 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     5101 tttaatgcct ttgtatcatg cgttaactaa acttgtttat tgcagcttat aatggttaca
     5161 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     5221 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gaattgactc aaatgatgtc
     5281 aattagtcta tcagaagctc atctggtctc ccttccgggg gacaagacat ccctgtttaa
     5341 tatttaaaca gcagtgttcc caaactgggt tcttatatcc cttgctctgg tcaaccaggt
     5401 tgcagggttt cctgtcctca caggaacgaa gtccctaaag aaacagtggc agccaggttt
     5461 agccccggaa ttgactggat tcctttttta gggcccattg gtatggcttt ttccccgtat
     5521 ccccccaggt gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg
     5581 tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga
     5641 gcgccggacc ggagcggagc cccgggcggc tcgctgctgc cccctagcgg gggagggacg
     5701 taattacatc cctgggggct ttgggggggg gctgtccctg atatctataa caagaaaata
     5761 tatatataat aagttatcac gtaagtagaa catgaaataa caatataatt atcgtatgag
     5821 ttaaatctta aaagtcacgt aaaagataat catgcgtcat tttgactcac gcggtcgtta
     5881 tagttcaaaa tcagtgacac ttaccgcatt gacaagcacg cctcacggga gctccaagcg
     5941 gcgactgaga tgtcctaaat gcacagcgac ggattcgcgc tatttagaaa gagagagcaa
     6001 tatttcaaga atgcatgcgt caattttacg cagactatct ttctagggtt aatctagctg
     6061 catcaggatc atatcgtcgg gtcttttttc cggctcagtc atcgcccaag ctggcgctat
     6121 ctgggcatcg gggaggaaga agcccgtgcc ttttcccgcg aggttgaagc ggcatggaaa
     6181 gagtttgccg aggatgactg ctgctgcatt gacgttgagc gaaaacgcac gtttaccatg
     6241 atgattcggg aaggtgtggc catgcacgcc tttaacggtg aactgttcgt tcaggccacc
     6301 tgggatacca gttcgtcgcg gcttttccgg acacagttcc ggatggtcag cccgaagcgc
     6361 atcagcaacc cgaacaatac cggcgacagc cggaactgcc gtgccggtgt gcagattaat
     6421 gacagcggtg cggcgctggg atattacgtc agcgaggacg ggtatcctgg ctggatgccg
     6481 cagaaatgga catggatacc ccgtgagtta cccggcgggc gcgcttggcg taatcatggt
     6541 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     6601 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     6661 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagct
//
