LOCUS       sequence-267617-        7468 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 7468)
  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 7468)
  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..7468
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1888..2262
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="PEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVL
                     KQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPG
                     ELAKHAVSEGTKAITKYTSAK"
     regulatory      2275..2284
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2281..2283,2284..2286,2287..3000)
                     /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(2281..2283,2284..2286,2287..2997)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2326..2347)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2587..2606)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2934..2955
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3073..3207
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3097..3116)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3151..3170
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3986..4048
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4178..4195)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4178..4194)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4187..4209)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4336..4791
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4423..4442)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4633..4654
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4817..4921
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4922..4990,4991..5782)
                     /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(5140..5159)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5953..6541
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6442..6461
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6695..6712
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6829..6850
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6865..6870,6871..6888,6889..6895)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6903..6919
                     /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     6908..6930
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6927..6943
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6927..6943
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   7434..7468
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 cattcttgaa atattgctct ctctttctaa atagcgcgaa tccgtcgctg tgcatttagg
       61 acatctcagt cgccgcttgg agctcccgtg aggcgtgctt gtcaatgcgg taagtgtcac
      121 tgattttgaa ctataacgac cgcgtgagtc aaaatgacgc atgattatct tttacgtgac
      181 ttttaagatt taactcatac gataattata ttgttatttc atgttctact tacgtgataa
      241 cttattatat atatattttc ttgttataga tctgtggaat gtgtgctaga gggacagccc
      301 ccccccaaag cccccaggga tgtaattacg tccctccccc gctagggggc agcagcgagc
      361 cgcccggggc tccgctccgg tccggcgctc cccccgcatc cccgagccgg cagcgtgcgg
      421 ggacagcccg ggcacgggga aggtggcacg ggatcgcttt cctctgaacg cttctcgctg
      481 ctctttgagc ctgcagacac ctggggggat acggggaaaa ggcctccaag gcctgcagga
      541 gtcaatggga aaaacccatt ggagccaagt acactgactc aatagggact ttccattggg
      601 ttttgcccag tacataaggt caataggggg tgagtcaaca ggaaagtccc attggagcca
      661 agtacattga gtcaataggg actttccaat gggttttgcc cagtacataa ggtcaatggg
      721 aggtaagcca atgggttttt cccattactg acatgtatac tgagtcatta gggactttcc
      781 aatgggtttt gcccagtaca taaggtcaat aggggtgaat caacaggaaa gtcccattgg
      841 agccaagtac actgagtcaa tagggacttt ccattgggtt ttgcccagta caaaaggtca
      901 atagggggtg agtcaatggg tttttcccat tattggcaca tacataaggt caataggggt
      961 gactagtgga gaagagcatg cttgagggct gagtgcccct cagtgggcag agagcacatg
     1021 gcccacagtc cctgagaagt tggggggagg ggtgggcaat tgaactggtg cctagagaag
     1081 gtggggcttg ggtaaactgg gaaagtgatg tggtgtactg gctccacctt tttccccagg
     1141 gtgggggaga accatatata agtgcagtag tctctgtgaa cattcggttt agtgaacctg
     1201 caggtcggcc gccacgaccg gtgccaccat gactgaatac aaaccaactg ttcgcctggc
     1261 aactcgtgat gatgttccac gtgcagttcg caccctggct gctgcatttg ctgactaccc
     1321 tgcaacccgt cacactgtgg acccagaccg ccacattgaa cgtgtgactg aactgcagga
     1381 gctgttcctg acccgtgtgg gcctggacat tggcaaagtg tgggtggcag atgatggtgc
     1441 tgctgtggca gtgtggacca cccctgaatc tgttgaagct ggtgcagtgt ttgctgagat
     1501 tggcccacgc atggcagaac tgtctggcag ccgcctggca gcacaacagc agatggaagg
     1561 tctgctggca ccacaccgcc caaaagaacc tgcttggttc ctggcaactg tgggtgtgag
     1621 ccctgaccac cagggtaagg gcctgggctc tgcagtggtg ctgcctggtg tggaagcagc
     1681 tgaacgtgca ggtgtgcctg ctttcctgga gacctcagct ccacgcaacc tgcctttcta
     1741 tgaacgcctg ggcttcactg tgactgctga tgtggaagtg ccagaaggcc cacgcacttg
     1801 gtgcatgact cgcaaaccag gtgctaggtg accgcaataa aaagacagaa taaaacgcac
     1861 gggtgttggg tcgtttgttc actcggccca gagccagcga agtctgctcc cgccccgaaa
     1921 aagggctcca agaaggcggt gactaaggcg cagaagaaag gcggcaagaa gcgcaagcgc
     1981 agccgcaagg agagctattc catctatgtg tacaaggttc tgaagcaggt ccaccctgac
     2041 accggcattt cgtccaaggc catgggcatc atgaattcgt ttgtgaacga cattttcgag
     2101 cgcatcgcag gtgaggcttc ccgcctggcg cattacaaca agcgctcgac catcacctcc
     2161 agggagatcc agacggccgt gcgcctgctg ctgcctgggg agttggccaa gcacgccgtg
     2221 tccgagggta ctaaggccat caccaagtac accagcgcta aggatccacc ggtcgccacc
     2281 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     2341 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     2401 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     2461 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     2521 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     2581 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     2641 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     2701 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     2761 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     2821 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     2881 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     2941 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
     3001 agcggccgcg actccagttc gtggccgtcg atcgtttaaa gggaggtagt gagtcgagat
     3061 atcgatacta gtgatccaga catgataaga tacattgatg agtttggaca aaccacaact
     3121 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     3181 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     3241 gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtatg
     3301 gctgattatg atcctctaga gggacagccc ccccccaaag cccccaggga tgtaattacg
     3361 tccctccccc gctagggggc agcagcgagc cgcccggggc tccgctccgg tccggcgctc
     3421 cccccgcatc cccgagccgg cagcgtgcgg ggacagcccg ggcacgggga aggtggcacg
     3481 ggatcgcttt cctctgaacg cttctcgctg ctctttgagc ctgcagacac ctggggggat
     3541 acggggaaaa agccatacca atggagctca cggggacagc ccccccccaa agcccccagg
     3601 gatgtaatta cgtccctccc ccgctagggg gcagcagcga gccgcccggg gctccgctcc
     3661 ggtccggcgc tccccccgca tccccgagcc ggcagcgtgc ggggacagcc cgggcacggg
     3721 gaaggtggca cgggatcgct ttcctctgaa cgcttctcgc tgctctttga gcctgcagac
     3781 acctgggggg atacggggaa aaagaattcg ataaaagttt tgttacttta tagaagaaat
     3841 tttgagtttt tgtttttttt aataaataaa taaacataaa taaattgttt gttgaattta
     3901 ttattagtat gtaagtgtaa atataataaa acttaatatc tattcaaatt aataaataaa
     3961 cctcgatata cagaccgata aaacacatgc gtcaatttta cacatgatta tctttaacgt
     4021 acgtcacaat atgattatct ttctagggtt aatctagctg cgtgttctgc agcgtgtcga
     4081 gcatcttcat ctgctccatc acgctgtaaa acacatttgc accgcgagtc tgcccgtcct
     4141 ccacgggttc aaaaacgtga atgaacgagg cgcgctcact ggccgtcgtt ttacaacgtc
     4201 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     4261 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     4321 tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     4381 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     4441 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     4501 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     4561 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     4621 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     4681 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     4741 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     4801 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     4861 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     4921 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     4981 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5041 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5101 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5161 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     5221 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     5281 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     5341 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     5401 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     5461 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     5521 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     5581 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     5641 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     5701 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     5761 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     5821 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     5881 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     5941 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6001 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6061 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     6121 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6181 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6241 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6301 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6361 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     6421 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     6481 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     6541 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     6601 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     6661 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     6721 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     6781 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     6841 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     6901 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
     6961 cgcccgccgg gtaactcacg gggtatccat gtccatttct gcggcatcca gccaggatac
     7021 ccgtcctcgc tgacgtaata tcccagcgcc gcaccgctgt cattaatctg cacaccggca
     7081 cggcagttcc ggctgtcgcc ggtattgttc gggttgctga tgcgcttcgg gctgaccatc
     7141 cggaactgtg tccggaaaag ccgcgacgaa ctggtatccc aggtggcctg aacgaacagt
     7201 tcaccgttaa aggcgtgcat ggccacacct tcccgaatca tcatggtaaa cgtgcgtttt
     7261 cgctcaacgt caatgcagca gcagtcatcc tcggcaaact ctttccatgc cgcttcaacc
     7321 tcgcgggaaa aggcacgggc ttcttcctcc ccgatgccca gatagcgcca gcttgggcga
     7381 tgactgagcc ggaaaaaaga cccgacgata tgatcctgat gcagctagat taaccctaga
     7441 aagatagtct gcgtaaaatt gacgcatg
//
