LOCUS       sequence-256386-        7142 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 7142)
  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 7142)
  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-CPspaH2B
FEATURES             Location/Qualifiers
     source          1..7142
                     /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)"
     enhancer        392..695
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        696..899
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     850..870
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1602..1976
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="PEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVL
                     KQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPG
                     ELAKHAVSEGTKAITKYTSAK"
     regulatory      1989..1998
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1995..1997,1998..2000,2001..2714)
                     /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(1995..1997,1998..2000,2001..2711)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2040..2061)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2301..2320)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2648..2669
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2787..2921
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2811..2830)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2865..2884
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3699..3761
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3891..3908)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3891..3907)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3900..3922)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4049..4504
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4136..4155)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4346..4367
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4530..4634
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4635..4703,4704..5495)
                     /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(4853..4872)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5666..6254
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6155..6174
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6408..6425
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6542..6563
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6578..6583,6584..6601,6602..6608)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6616..6632
                     /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     6621..6643
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6640..6656
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6640..6656
                     /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 tgtgctagca ttagttatta atagtaatca attacggggt
      361 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
      421 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
      481 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
      541 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
      601 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
      661 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
      721 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
      781 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
      841 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg
      901 gtttagtgaa cctgcaggtc ggccgccacg accggtgcca ccatgactga atacaaacca
      961 actgttcgcc tggcaactcg tgatgatgtt ccacgtgcag ttcgcaccct ggctgctgca
     1021 tttgctgact accctgcaac ccgtcacact gtggacccag accgccacat tgaacgtgtg
     1081 actgaactgc aggagctgtt cctgacccgt gtgggcctgg acattggcaa agtgtgggtg
     1141 gcagatgatg gtgctgctgt ggcagtgtgg accacccctg aatctgttga agctggtgca
     1201 gtgtttgctg agattggccc acgcatggca gaactgtctg gcagccgcct ggcagcacaa
     1261 cagcagatgg aaggtctgct ggcaccacac cgcccaaaag aacctgcttg gttcctggca
     1321 actgtgggtg tgagccctga ccaccagggt aagggcctgg gctctgcagt ggtgctgcct
     1381 ggtgtggaag cagctgaacg tgcaggtgtg cctgctttcc tggagacctc agctccacgc
     1441 aacctgcctt tctatgaacg cctgggcttc actgtgactg ctgatgtgga agtgccagaa
     1501 ggcccacgca cttggtgcat gactcgcaaa ccaggtgcta ggtgaccgca ataaaaagac
     1561 agaataaaac gcacgggtgt tgggtcgttt gttcactcgg cccagagcca gcgaagtctg
     1621 ctcccgcccc gaaaaagggc tccaagaagg cggtgactaa ggcgcagaag aaaggcggca
     1681 agaagcgcaa gcgcagccgc aaggagagct attccatcta tgtgtacaag gttctgaagc
     1741 aggtccaccc tgacaccggc atttcgtcca aggccatggg catcatgaat tcgtttgtga
     1801 acgacatttt cgagcgcatc gcaggtgagg cttcccgcct ggcgcattac aacaagcgct
     1861 cgaccatcac ctccagggag atccagacgg ccgtgcgcct gctgctgcct ggggagttgg
     1921 ccaagcacgc cgtgtccgag ggtactaagg ccatcaccaa gtacaccagc gctaaggatc
     1981 caccggtcgc caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     2041 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     2101 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     2161 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     2221 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     2281 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     2341 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     2401 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     2461 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     2521 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     2581 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     2641 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     2701 agctgtacaa gtaaagcggc cgcgactcca gttcgtggcc gtcgatcgtt taaagggagg
     2761 tagtgagtcg agatatcgat actagtgatc cagacatgat aagatacatt gatgagtttg
     2821 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     2881 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     2941 attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct
     3001 acaaatgtgg tatggctgat tatgatcctc tagagggaca gccccccccc aaagccccca
     3061 gggatgtaat tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct
     3121 ccggtccggc gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg
     3181 gggaaggtgg cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag
     3241 acacctgggg ggatacgggg aaaagccata ccaatggagc tcacggggac agcccccccc
     3301 caaagccccc agggatgtaa ttacgtccct cccccgctag ggggcagcag cgagccgccc
     3361 ggggctccgc tccggtccgg cgctcccccc gcatccccga gccggcagcg tgcggggaca
     3421 gcccgggcac ggggaaggtg gcacgggatc gctttcctct gaacgcttct cgctgctctt
     3481 tgagcctgca gacacctggg gggatacggg gaaaaagaat tcgataaaag ttttgttact
     3541 ttatagaaga aattttgagt ttttgttttt tttaataaat aaataaacat aaataaattg
     3601 tttgttgaat ttattattag tatgtaagtg taaatataat aaaacttaat atctattcaa
     3661 attaataaat aaacctcgat atacagaccg ataaaacaca tgcgtcaatt ttacacatga
     3721 ttatctttaa cgtacgtcac aatatgatta tctttctagg gttaatctag ctgcgtgttc
     3781 tgcagcgtgt cgagcatctt catctgctcc atcacgctgt aaaacacatt tgcaccgcga
     3841 gtctgcccgt cctccacggg ttcaaaaacg tgaatgaacg aggcgcgctc actggccgtc
     3901 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     3961 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     4021 cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     4081 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     4141 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     4201 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     4261 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     4321 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     4381 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     4441 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     4501 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4561 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     4621 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     4681 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     4741 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     4801 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     4861 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     4921 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     4981 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5041 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5101 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5161 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     5221 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     5281 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     5341 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5401 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5461 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     5521 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     5581 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5641 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5701 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     5761 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     5821 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     5881 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5941 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6001 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6061 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6121 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6181 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     6241 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6301 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     6361 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     6421 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     6481 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     6541 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     6601 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     6661 attacgccaa gcgcgcccgc cgggtaactc acggggtatc catgtccatt tctgcggcat
     6721 ccagccagga tacccgtcct cgctgacgta atatcccagc gccgcaccgc tgtcattaat
     6781 ctgcacaccg gcacggcagt tccggctgtc gccggtattg ttcgggttgc tgatgcgctt
     6841 cgggctgacc atccggaact gtgtccggaa aagccgcgac gaactggtat cccaggtggc
     6901 ctgaacgaac agttcaccgt taaaggcgtg catggccaca ccttcccgaa tcatcatggt
     6961 aaacgtgcgt tttcgctcaa cgtcaatgca gcagcagtca tcctcggcaa actctttcca
     7021 tgccgcttca acctcgcggg aaaaggcacg ggcttcttcc tccccgatgc ccagatagcg
     7081 ccagcttggg cgatgactga gccggaaaaa agacccgacg atatgatcct gatgcagcta
     7141 ga
//
