LOCUS       sequence-256387-        7378 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 7378)
  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 7378)
  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..7378
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   5..39
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     CDS             1931..2950
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             1931..2950
                     /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    3023..3157
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3047..3066)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3101..3120
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3935..3997
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4127..4144)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4127..4143)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4136..4158)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4285..4740
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4372..4391)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4582..4603
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4766..4870
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4871..4939,4940..5731)
                     /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(5089..5108)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5902..6490
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6391..6410
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6644..6661
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6778..6799
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6814..6819,6820..6837,6838..6844)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6852..6868
                     /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     6857..6879
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6876..6892
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6876..6892
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttaaccctag aaagatagtc tgcgtaaaat tgacgcatgc attcttgaaa tattgctctc
       61 tctttctaaa tagcgcgaat ccgtcgctgt gcatttagga catctcagtc gccgcttgga
      121 gctcccgtga ggcgtgcttg tcaatgcggt aagtgtcact gattttgaac tataacgacc
      181 gcgtgagtca aaatgacgca tgattatctt ttacgtgact tttaagattt aactcatacg
      241 ataattatat tgttatttca tgttctactt acgtgataac ttattatata tatattttct
      301 tgttatagat ctgtggaatg tgtgctagag ggacagcccc cccccaaagc ccccagggat
      361 gtaattacgt ccctcccccg ctagggggca gcagcgagcc gcccggggct ccgctccggt
      421 ccggcgctcc ccccgcatcc ccgagccggc agcgtgcggg gacagcccgg gcacggggaa
      481 ggtggcacgg gatcgctttc ctctgaacgc ttctcgctgc tctttgagcc tgcagacacc
      541 tggggggata cggggaaaag gcctccaagg cctgcaggag tcaatgggaa aaacccattg
      601 gagccaagta cactgactca atagggactt tccattgggt tttgcccagt acataaggtc
      661 aatagggggt gagtcaacag gaaagtccca ttggagccaa gtacattgag tcaataggga
      721 ctttccaatg ggttttgccc agtacataag gtcaatggga ggtaagccaa tgggtttttc
      781 ccattactga catgtatact gagtcattag ggactttcca atgggttttg cccagtacat
      841 aaggtcaata ggggtgaatc aacaggaaag tcccattgga gccaagtaca ctgagtcaat
      901 agggactttc cattgggttt tgcccagtac aaaaggtcaa tagggggtga gtcaatgggt
      961 ttttcccatt attggcacat acataaggtc aataggggtg actagtggag aagagcatgc
     1021 ttgagggctg agtgcccctc agtgggcaga gagcacatgg cccacagtcc ctgagaagtt
     1081 ggggggaggg gtgggcaatt gaactggtgc ctagagaagg tggggcttgg gtaaactggg
     1141 aaagtgatgt ggtgtactgg ctccaccttt ttccccaggg tgggggagaa ccatatataa
     1201 gtgcagtagt ctctgtgaac attcggttta gtgaacctgc aggtcggccg ccacgaccgg
     1261 tgccaccatg actgaataca aaccaactgt tcgcctggca actcgtgatg atgttccacg
     1321 tgcagttcgc accctggctg ctgcatttgc tgactaccct gcaacccgtc acactgtgga
     1381 cccagaccgc cacattgaac gtgtgactga actgcaggag ctgttcctga cccgtgtggg
     1441 cctggacatt ggcaaagtgt gggtggcaga tgatggtgct gctgtggcag tgtggaccac
     1501 ccctgaatct gttgaagctg gtgcagtgtt tgctgagatt ggcccacgca tggcagaact
     1561 gtctggcagc cgcctggcag cacaacagca gatggaaggt ctgctggcac cacaccgccc
     1621 aaaagaacct gcttggttcc tggcaactgt gggtgtgagc cctgaccacc agggtaaggg
     1681 cctgggctct gcagtggtgc tgcctggtgt ggaagcagct gaacgtgcag gtgtgcctgc
     1741 tttcctggag acctcagctc cacgcaacct gcctttctat gaacgcctgg gcttcactgt
     1801 gactgctgat gtggaagtgc cagaaggccc acgcacttgg tgcatgactc gcaaaccagg
     1861 tgctaggtga ccgcaataaa aagacagaat aaaacgcacg ggtgttgggt cgtttgttca
     1921 ctcggcaata aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt
     1981 cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt
     2041 cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa
     2101 agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga
     2161 cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac
     2221 gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat
     2281 ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca
     2341 aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt
     2401 gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga
     2461 tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt
     2521 cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga
     2581 ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt
     2641 ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc
     2701 gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt
     2761 tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc
     2821 cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga
     2881 tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc
     2941 aaaggaatag agcggccgcg actccagttc gtggccgtcg atcgtttaaa gggaggtagt
     3001 gagtcgagat atcgatacta gtgatccaga catgataaga tacattgatg agtttggaca
     3061 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
     3121 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
     3181 tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa
     3241 atgtggtatg gctgattatg atcctctaga gggacagccc ccccccaaag cccccaggga
     3301 tgtaattacg tccctccccc gctagggggc agcagcgagc cgcccggggc tccgctccgg
     3361 tccggcgctc cccccgcatc cccgagccgg cagcgtgcgg ggacagcccg ggcacgggga
     3421 aggtggcacg ggatcgcttt cctctgaacg cttctcgctg ctctttgagc ctgcagacac
     3481 ctggggggat acggggaaaa gccataccaa tggagctcac ggggacagcc cccccccaaa
     3541 gcccccaggg atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg
     3601 ctccgctccg gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc
     3661 gggcacgggg aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag
     3721 cctgcagaca cctgggggga tacggggaaa aagaattcga taaaagtttt gttactttat
     3781 agaagaaatt ttgagttttt gtttttttta ataaataaat aaacataaat aaattgtttg
     3841 ttgaatttat tattagtatg taagtgtaaa tataataaaa cttaatatct attcaaatta
     3901 ataaataaac ctcgatatac agaccgataa aacacatgcg tcaattttac acatgattat
     3961 ctttaacgta cgtcacaata tgattatctt tctagggtta atctagctgc gtgttctgca
     4021 gcgtgtcgag catcttcatc tgctccatca cgctgtaaaa cacatttgca ccgcgagtct
     4081 gcccgtcctc cacgggttca aaaacgtgaa tgaacgaggc gcgctcactg gccgtcgttt
     4141 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     4201 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     4261 tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     4321 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     4381 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     4441 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     4501 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     4561 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     4621 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     4681 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     4741 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     4801 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     4861 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     4921 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     4981 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     5041 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     5101 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     5161 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     5221 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     5281 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     5341 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     5401 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     5461 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     5521 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     5581 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     5641 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     5701 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     5761 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     5821 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     5881 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     5941 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     6001 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     6061 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     6121 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     6181 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     6241 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     6301 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     6361 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     6421 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     6481 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     6541 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     6601 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     6661 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     6721 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     6781 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     6841 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     6901 cgccaagcgc gcccgccggg taactcacgg ggtatccatg tccatttctg cggcatccag
     6961 ccaggatacc cgtcctcgct gacgtaatat cccagcgccg caccgctgtc attaatctgc
     7021 acaccggcac ggcagttccg gctgtcgccg gtattgttcg ggttgctgat gcgcttcggg
     7081 ctgaccatcc ggaactgtgt ccggaaaagc cgcgacgaac tggtatccca ggtggcctga
     7141 acgaacagtt caccgttaaa ggcgtgcatg gccacacctt cccgaatcat catggtaaac
     7201 gtgcgttttc gctcaacgtc aatgcagcag cagtcatcct cggcaaactc tttccatgcc
     7261 gcttcaacct cgcgggaaaa ggcacgggct tcttcctccc cgatgcccag atagcgccag
     7321 cttgggcgat gactgagccg gaaaaaagac ccgacgatat gatcctgatg cagctaga
//
