LOCUS       sequence-269198-        7379 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Piggybac vector for SRIRACCHA-mediated mutation enrichment 
            (surrogate target for HDR).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7379)
  AUTHORS   Wen Y, Liao G, Pritchard T, Zhao TT, Connelly JP, Pruett-Miller SM, 
            Blanc V, Davidson NO, Madison BB
  TITLE     A stable but reversible integrated surrogate reporter for assaying 
            CRISPR/Cas9-stimulated homology-directed repair.
  JOURNAL   J Biol Chem. 2017 Apr 14;292(15):6148-6162. doi: 
            10.1074/jbc.M117.777722. Epub 2017 Feb 22.
  PUBMED    28228480
REFERENCE   2  (bases 1 to 7379)
  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: 
            "10.1074/jbc.M117.777722"; journalName: "J Biol Chem"; date: 
            "2017-04-14- 14"; volume: "292"; issue: "15"; pages: "6148-6162"
COMMENT     Sequence Label: BII-C3H
FEATURES             Location/Qualifiers
     source          1..7379
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..24,25..31)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    39..55
                     /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     44..66
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     63..79
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     63..79
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   570..604
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     CDS             2496..3515
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             2496..3515
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     polyA_signal    3588..3722
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3612..3631)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3666..3685
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4501..4563
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4693..4710)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4693..4709)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4702..4724)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4851..5306
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4938..4957)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5148..5169
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5332..5436
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5437..5505,5506..6297)
                     /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(5655..5674)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6468..7056
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6957..6976
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7210..7227
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7344..7365
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
       61 cacaggaaac agctatgacc atgattacgc caagcgcgcc cgccgggtaa ctcacggggt
      121 atccatgtcc atttctgcgg catccagcca ggatacccgt cctcgctgac gtaatatccc
      181 agcgccgcac cgctgtcatt aatctgcaca ccggcacggc agttccggct gtcgccggta
      241 ttgttcgggt tgctgatgcg cttcgggctg accatccgga actgtgtccg gaaaagccgc
      301 gacgaactgg tatcccaggt ggcctgaacg aacagttcac cgttaaaggc gtgcatggcc
      361 acaccttccc gaatcatcat ggtaaacgtg cgttttcgct caacgtcaat gcagcagcag
      421 tcatcctcgg caaactcttt ccatgccgct tcaacctcgc gggaaaaggc acgggcttct
      481 tcctccccga tgcccagata gcgccagctt gggcgatgac tgagccggaa aaaagacccg
      541 acgatatgat cctgatgcag ctagattaac cctagaaaga tagtctgcgt aaaattgacg
      601 catgcattct tgaaatattg ctctctcttt ctaaatagcg cgaatccgtc gctgtgcatt
      661 taggacatct cagtcgccgc ttggagctcc cgtgaggcgt gcttgtcaat gcggtaagtg
      721 tcactgattt tgaactataa cgaccgcgtg agtcaaaatg acgcatgatt atcttttacg
      781 tgacttttaa gatttaactc atacgataat tatattgtta tttcatgttc tacttacgtg
      841 ataacttatt atatatatat tttcttgtta tagatctgtg gaatgtgtgc tagagggaca
      901 gccccccccc aaagccccca gggatgtaat tacgtccctc ccccgctagg gggcagcagc
      961 gagccgcccg gggctccgct ccggtccggc gctccccccg catccccgag ccggcagcgt
     1021 gcggggacag cccgggcacg gggaaggtgg cacgggatcg ctttcctctg aacgcttctc
     1081 gctgctcttt gagcctgcag acacctgggg ggatacgggg aaaaggcctc caaggcctgc
     1141 aggagtcaat gggaaaaacc cattggagcc aagtacactg actcaatagg gactttccat
     1201 tgggttttgc ccagtacata aggtcaatag ggggtgagtc aacaggaaag tcccattgga
     1261 gccaagtaca ttgagtcaat agggactttc caatgggttt tgcccagtac ataaggtcaa
     1321 tgggaggtaa gccaatgggt ttttcccatt actgacatgt atactgagtc attagggact
     1381 ttccaatggg ttttgcccag tacataaggt caataggggt gaatcaacag gaaagtccca
     1441 ttggagccaa gtacactgag tcaataggga ctttccattg ggttttgccc agtacaaaag
     1501 gtcaataggg ggtgagtcaa tgggtttttc ccattattgg cacatacata aggtcaatag
     1561 gggtgactag tggagaagag catgcttgag ggctgagtgc ccctcagtgg gcagagagca
     1621 catggcccac agtccctgag aagttggggg gaggggtggg caattgaact ggtgcctaga
     1681 gaaggtgggg cttgggtaaa ctgggaaagt gatgtggtgt actggctcca cctttttccc
     1741 cagggtgggg gagaaccata tataagtgca gtagtctctg tgaacattcg gtttagtgaa
     1801 cctgcaggtc ggccgccacg accggtgcca ccatgactga atacaaacca actgttcgcc
     1861 tggcaactcg tgatgatgtt ccacgtgcag ttcgcaccct ggctgctgca tttgctgact
     1921 accctgcaac ccgtcacact gtggacccag accgccacat tgaacgtgtg actgaactgc
     1981 aggagctgtt cctgacccgt gtgggcctgg acattggcaa agtgtgggtg gcagatgatg
     2041 gtgctgctgt ggcagtgtgg accacccctg aatctgttga agctggtgca gtgtttgctg
     2101 agattggccc acgcatggca gaactgtctg gcagccgcct ggcagcacaa cagcagatgg
     2161 aaggtctgct ggcaccacac cgcccaaaag aacctgcttg gttcctggca actgtgggtg
     2221 tgagccctga ccaccagggt aagggcctgg gctctgcagt ggtgctgcct ggtgtggaag
     2281 cagctgaacg tgcaggtgtg cctgctttcc tggagacctc agctccacgc aacctgcctt
     2341 tctatgaacg cctgggcttc actgtgactg ctgatgtgga agtgccagaa ggcccacgca
     2401 cttggtgcat gactcgcaaa ccaggtgcta ggtgaccgca ataaaaagac agaataaaac
     2461 gcacgggtgt tgggtcgttt gttcactcgg caataaagcc tgaactcacc gcgacgtctg
     2521 tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg
     2581 gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa
     2641 atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg
     2701 cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg acctattgca
     2761 tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg
     2821 ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga
     2881 gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca
     2941 tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca
     3001 gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag
     3061 tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca
     3121 taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca
     3181 acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc
     3241 ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc
     3301 ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg
     3361 gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc
     3421 gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc
     3481 gacgccccag cactcgtccg agggcaaagg aatagagcgg ccgcgactcc agttcgtggc
     3541 cgtcgatcgt ttaaagggag gtagtgagtc gagatatcga tactagtgat ccagacatga
     3601 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta
     3661 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag
     3721 ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt
     3781 tttaaagcaa gtaaaacctc tacaaatgtg gtatggctga ttatgatcct ctagagggac
     3841 agcccccccc caaagccccc agggatgtaa ttacgtccct cccccgctag ggggcagcag
     3901 cgagccgccc ggggctccgc tccggtccgg cgctcccccc gcatccccga gccggcagcg
     3961 tgcggggaca gcccgggcac ggggaaggtg gcacgggatc gctttcctct gaacgcttct
     4021 cgctgctctt tgagcctgca gacacctggg gggatacggg gaaaaagcca taccaatgga
     4081 gctcacgggg acagcccccc cccaaagccc ccagggatgt aattacgtcc ctcccccgct
     4141 agggggcagc agcgagccgc ccggggctcc gctccggtcc ggcgctcccc ccgcatcccc
     4201 gagccggcag cgtgcgggga cagcccgggc acggggaagg tggcacggga tcgctttcct
     4261 ctgaacgctt ctcgctgctc tttgagcctg cagacacctg gggggatacg gggaaaaaga
     4321 attcgataaa agttttgtta ctttatagaa gaaattttga gtttttgttt tttttaataa
     4381 ataaataaac ataaataaat tgtttgttga atttattatt agtatgtaag tgtaaatata
     4441 ataaaactta atatctattc aaattaataa ataaacctcg atatacagac cgataaaaca
     4501 catgcgtcaa ttttacacat gattatcttt aacgtacgtc acaatatgat tatctttcta
     4561 gggttaatct agctgcgtgt tctgcagcgt gtcgagcatc ttcatctgct ccatcacgct
     4621 gtaaaacaca tttgcaccgc gagtctgccc gtcctccacg ggttcaaaaa cgtgaatgaa
     4681 cgaggcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     4741 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     4801 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg
     4861 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     4921 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     4981 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     5041 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     5101 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     5161 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     5221 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     5281 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     5341 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     5401 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     5461 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     5521 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     5581 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     5641 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     5701 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     5761 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     5821 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     5881 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     5941 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     6001 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     6061 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     6121 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     6181 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     6241 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     6301 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     6361 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     6421 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6481 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6541 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6601 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6661 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     6721 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     6781 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6841 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6901 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6961 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     7021 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     7081 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     7141 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     7201 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     7261 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     7321 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggc
//
