LOCUS       sequence-350235-        6945 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac transposon containing hU6 promotor and pegRNA cloning site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6945)
  AUTHORS   Wolff JH, Haldrup J, Thomsen EA, Andersen S, Mikkelsen JG
  TITLE     piggyPrime: High-Efficacy Prime Editing in Human Cells Using 
            piggyBac-Based DNA Transposition.
  JOURNAL   Front Genome Ed. 2021 Nov 12;3:786893. doi: 
            10.3389/fgeed.2021.786893. eCollection 2021.
  PUBMED    34870275
REFERENCE   2  (bases 1 to 6945)
  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: "Front 
            Genome Ed."; date: "2021-11-12"; volume: "3:786893. doi"; pages: " 
            10.3389/fgeed.2021.786893. eCollection 2021"
COMMENT     Sequence Label: pPBT-peRNA_GG-Puro
FEATURES             Location/Qualifiers
     source          1..6945
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        79..183
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(184..252,253..1044)
                     /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(402..421)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1215..1803
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1704..1723
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1957..1974
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2091..2112
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2127..2132,2133..2150,2151..2157)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2165..2181
                     /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)."
     primer_bind     2170..2192
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2189..2205
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2189..2205
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   2696..2730
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        3016..3264
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3016..3036
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3187..3206
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     terminator      complement(3274..3305)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(3322..3999)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     RBS             4006..4017
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    4035..4053
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4060..4078
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        4199..5377
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4430..5368
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     5325..5345
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             5390..5989
                     /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(5390..5409)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5886..5906
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     repeat_region   6193..6255
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6385..6402)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6385..6401)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6394..6416)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      join(6543..6945,1..53)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6630..6649)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6840..6861
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
       61 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
      121 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
      181 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
      241 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
      301 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
      361 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
      421 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
      481 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
      541 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
      601 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
      661 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
      721 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
      781 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
      841 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
      901 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
      961 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     1021 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     1081 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     1141 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     1201 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     1261 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     1321 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     1381 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     1441 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     1501 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     1561 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     1621 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     1681 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     1741 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     1801 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     1861 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     1921 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     1981 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     2041 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
     2101 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
     2161 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag
     2221 cgcgcccgcc gggtaactca cggggtatcc atgtccattt ctgcggcatc cagccaggat
     2281 acccgtcctc gctgacgtaa tatcccagcg ccgcaccgct gtcattaatc tgcacaccgg
     2341 cacggcagtt ccggctgtcg ccggtattgt tcgggttgct gatgcgcttc gggctgacca
     2401 tccggaactg tgtccggaaa agccgcgacg aactggtatc ccaggtggcc tgaacgaaca
     2461 gttcaccgtt aaaggcgtgc atggccacac cttcccgaat catcatggta aacgtgcgtt
     2521 ttcgctcaac gtcaatgcag cagcagtcat cctcggcaaa ctctttccat gccgcttcaa
     2581 cctcgcggga aaaggcacgg gcttcttcct ccccgatgcc cagatagcgc cagcttgggc
     2641 gatgactgag ccggaaaaaa gacccgacga tatgatcctg atgcagctag attaacccta
     2701 gaaagatagt ctgcgtaaaa ttgacgcatg cattcttgaa atattgctct ctctttctaa
     2761 atagcgcgaa tccgtcgctg tgcatttagg acatctcagt cgccgcttgg agctcccgtg
     2821 aggcgtgctt gtcaatgcgg taagtgtcac tgattttgaa ctataacgac cgcgtgagtc
     2881 aaaatgacgc atgattatct tttacgtgac ttttaagatt taactcatac gataattata
     2941 ttgttatttc atgttctact tacgtgataa cttattatat atatattttc ttgttataga
     3001 tatcaagctt ccagagaggg cctatttccc atgattcctt catatttgca tatacgatac
     3061 aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa
     3121 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
     3181 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
     3241 atcttgtgga aaggacgaaa cacccgagac ggtagtcaaa agcctccggt cggaggcttt
     3301 tgacttggct gaggagtgcc attaagcacc ggtggagtga cgaccttcag cacgttcgta
     3361 ctgttcaacg atggtgtagt cttcgttgtg ggaggtgatg tccagtttga tgtcggtttt
     3421 gtaagcaccc ggcagctgaa ccggtttttt agccatgtag gtggttttaa cttcagcgtc
     3481 gtagtgacca ccgtctttca gtttcagacg cattttgatt tcacctttca gagcaccgtc
     3541 ttccgggtac atacgttcgg tggaagcttc ccaacccatg gtttttttct gcataaccgg
     3601 accgtcggac gggaagttgg taccacgcag tttaactttg tagatgaact caccgtcttg
     3661 cagggaggag tcctgggtaa cggtaacaac accaccgtct tcgaagttca taacacgttc
     3721 ccatttgaaa ccttccggga aggacagttt caggtagtcc gggatgtcag ccgggtgttt
     3781 aacgtaagct ttggaaccgt actggaactg cggggacagg atgtcccaag cgaacggcag
     3841 cggaccacct ttggtaactt tcagtttagc ggtctgggta ccttcgtacg gacgaccttc
     3901 accttcacct tcgatttcga actcgtgacc gttaacggaa ccttccatac gaactttgaa
     3961 acgcatgaac tctttgataa cgtcttcgct actcgccata gatcctttct cctctttcag
     4021 atccgtgctc agtatctcta tcactgatag ggatgtcaat ctctatcact gatagggacg
     4081 tctcgttttt ttaagcttgg cgtaactaga tccccgggtg caaagatgga taaagtttta
     4141 aacagagagg aatctttgca gctaatggac cttctaggtc ttgaaaggag tgggaattgg
     4201 ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg
     4261 aggggtcggc aattgatccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga
     4321 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt
     4381 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt
     4441 gtgtggttcc cgcgggcctg gcctctttac gggttatggc ccttgcgtgc cttgaattac
     4501 ttccacctgg ctgcagtacg tgattcttga tcccgagctt cgggttggaa gtgggtggga
     4561 gagttcgagg ccttgcgctt aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc
     4621 tgggcgctgg ggccgccgcg tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt
     4681 cgataagtct ctagccattt aaaatttttg atgacctgct gcgacgcttt ttttctggca
     4741 agatagtctt gtaaatgcgg gccaagatct gcacactggt atttcggttt ttggggccgc
     4801 gggcggcgac ggggcccgtg cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc
     4861 gcggccaccg agaatcggac gggggtagtc tcaagctggc cggcctgctc tggtgcctgg
     4921 cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt cggcaccagt
     4981 tgcgtgagcg gaaagatggc cgcttcccgg ccctgctgca gggagctcaa aatggaggac
     5041 gcggcgctcg ggagagcggg cgggtgagtc acccacacaa aggaaaaggg cctttccgtc
     5101 ctcagccgtc gcttcatgtg actccacgga gtaccgggcg ccgtccaggc acctcgatta
     5161 gttctcgagc ttttggagta cgtcgtcttt aggttggggg gaggggtttt atgcgatgga
     5221 gtttccccac actgagtggg tggagactga agttaggcca gcttggcact tgatgtaatt
     5281 ctccttggaa tttgcccttt ttgagtttgg atcttggttc attctcaagc ctcagacagt
     5341 ggttcaaagt ttttttcttc catttcaggt gtcgtgacgt acggccacca tgaccgagta
     5401 caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc
     5461 cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga
     5521 gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt
     5581 gtgggtcgcg gacgacggcg ccgccgtggc ggtctggacc acgccggaga gcgtcgaagc
     5641 gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc
     5701 cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt
     5761 cctggccacc gtcggagtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt
     5821 gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc
     5881 gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt
     5941 gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgaa cgcgttaacc
     6001 cgggctgcag gaattcgata aaagttttgt tactttatag aagaaatttt gagtttttgt
     6061 tttttttaat aaataaataa acataaataa attgtttgtt gaatttatta ttagtatgta
     6121 agtgtaaata taataaaact taatatctat tcaaattaat aaataaacct cgatatacag
     6181 accgataaaa cacatgcgtc aattttacac atgattatct ttaacgtacg tcacaatatg
     6241 attatctttc tagggttaat ctagctgcgt gttctgcagc gtgtcgagca tcttcatctg
     6301 ctccatcacg ctgtaaaaca catttgcacc gcgagtctgc ccgtcctcca cgggttcaaa
     6361 aacgtgaatg aacgaggcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa
     6421 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     6481 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     6541 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     6601 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     6661 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     6721 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     6781 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     6841 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     6901 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagct
//
