LOCUS       sequence-256388-        7467 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 7467)
  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 7467)
  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-C3H2B
FEATURES             Location/Qualifiers
     source          1..7467
                     /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             1927..2301
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="PEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVL
                     KQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPG
                     ELAKHAVSEGTKAITKYTSAK"
     regulatory      2314..2323
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2320..2322,2323..2325,2326..3039)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2320..2322,2323..2325,2326..3036)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2365..2386)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2626..2645)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2973..2994
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3112..3246
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3136..3155)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3190..3209
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4024..4086
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4216..4233)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4216..4232)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4225..4247)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4374..4829
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4461..4480)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4671..4692
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4855..4959
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4960..5028,5029..5820)
                     /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(5178..5197)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5991..6579
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6480..6499
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6733..6750
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6867..6888
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6903..6908,6909..6926,6927..6933)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6941..6957
                     /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     6946..6968
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6965..6981
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6965..6981
                     /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 ctcggcccag agccagcgaa gtctgctccc gccccgaaaa agggctccaa gaaggcggtg
     1981 actaaggcgc agaagaaagg cggcaagaag cgcaagcgca gccgcaagga gagctattcc
     2041 atctatgtgt acaaggttct gaagcaggtc caccctgaca ccggcatttc gtccaaggcc
     2101 atgggcatca tgaattcgtt tgtgaacgac attttcgagc gcatcgcagg tgaggcttcc
     2161 cgcctggcgc attacaacaa gcgctcgacc atcacctcca gggagatcca gacggccgtg
     2221 cgcctgctgc tgcctgggga gttggccaag cacgccgtgt ccgagggtac taaggccatc
     2281 accaagtaca ccagcgctaa ggatccaccg gtcgccacca tggtgagcaa gggcgaggag
     2341 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     2401 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     2461 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     2521 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     2581 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     2641 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     2701 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     2761 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     2821 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     2881 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     2941 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     3001 gccgggatca ctctcggcat ggacgagctg tacaagtaaa gcggccgcga ctccagttcg
     3061 tggccgtcga tcgtttaaag ggaggtagtg agtcgagata tcgatactag tgatccagac
     3121 atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc
     3181 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     3241 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag
     3301 gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga tcctctagag
     3361 ggacagcccc cccccaaagc ccccagggat gtaattacgt ccctcccccg ctagggggca
     3421 gcagcgagcc gcccggggct ccgctccggt ccggcgctcc ccccgcatcc ccgagccggc
     3481 agcgtgcggg gacagcccgg gcacggggaa ggtggcacgg gatcgctttc ctctgaacgc
     3541 ttctcgctgc tctttgagcc tgcagacacc tggggggata cggggaaaag ccataccaat
     3601 ggagctcacg gggacagccc ccccccaaag cccccaggga tgtaattacg tccctccccc
     3661 gctagggggc agcagcgagc cgcccggggc tccgctccgg tccggcgctc cccccgcatc
     3721 cccgagccgg cagcgtgcgg ggacagcccg ggcacgggga aggtggcacg ggatcgcttt
     3781 cctctgaacg cttctcgctg ctctttgagc ctgcagacac ctggggggat acggggaaaa
     3841 agaattcgat aaaagttttg ttactttata gaagaaattt tgagtttttg ttttttttaa
     3901 taaataaata aacataaata aattgtttgt tgaatttatt attagtatgt aagtgtaaat
     3961 ataataaaac ttaatatcta ttcaaattaa taaataaacc tcgatataca gaccgataaa
     4021 acacatgcgt caattttaca catgattatc tttaacgtac gtcacaatat gattatcttt
     4081 ctagggttaa tctagctgcg tgttctgcag cgtgtcgagc atcttcatct gctccatcac
     4141 gctgtaaaac acatttgcac cgcgagtctg cccgtcctcc acgggttcaa aaacgtgaat
     4201 gaacgaggcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     4261 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     4321 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc
     4381 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     4441 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     4501 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     4561 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     4621 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     4681 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     4741 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     4801 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     4861 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     4921 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     4981 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     5041 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     5101 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     5161 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     5221 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     5281 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     5341 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     5401 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     5461 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     5521 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     5581 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     5641 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     5701 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     5761 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     5821 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     5881 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     5941 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     6001 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6061 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6121 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     6181 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     6241 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     6301 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     6361 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     6421 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     6481 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     6541 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     6601 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     6661 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     6721 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     6781 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     6841 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     6901 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     6961 cacacaggaa acagctatga ccatgattac gccaagcgcg cccgccgggt aactcacggg
     7021 gtatccatgt ccatttctgc ggcatccagc caggataccc gtcctcgctg acgtaatatc
     7081 ccagcgccgc accgctgtca ttaatctgca caccggcacg gcagttccgg ctgtcgccgg
     7141 tattgttcgg gttgctgatg cgcttcgggc tgaccatccg gaactgtgtc cggaaaagcc
     7201 gcgacgaact ggtatcccag gtggcctgaa cgaacagttc accgttaaag gcgtgcatgg
     7261 ccacaccttc ccgaatcatc atggtaaacg tgcgttttcg ctcaacgtca atgcagcagc
     7321 agtcatcctc ggcaaactct ttccatgccg cttcaacctc gcgggaaaag gcacgggctt
     7381 cttcctcccc gatgcccaga tagcgccagc ttgggcgatg actgagccgg aaaaaagacc
     7441 cgacgatatg atcctgatgc agctaga
//
