LOCUS       sequence-267615-        7143 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 7143)
  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 7143)
  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..7143
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        353..656
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        657..860
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     811..831
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1563..1937
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="PEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVL
                     KQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPG
                     ELAKHAVSEGTKAITKYTSAK"
     regulatory      1950..1959
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1956..1958,1959..1961,1962..2675)
                     /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(1956..1958,1959..1961,1962..2672)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2001..2022)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2262..2281)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2609..2630
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2748..2882
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2772..2791)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2826..2845
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3661..3723
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3853..3870)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3853..3869)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3862..3884)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4011..4466
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4098..4117)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4308..4329
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4492..4596
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4597..4665,4666..5457)
                     /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(4815..4834)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5628..6216
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6117..6136
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6370..6387
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6504..6525
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6540..6545,6546..6563,6564..6570)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6578..6594
                     /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     6583..6605
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6602..6618
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6602..6618
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   7109..7143
                     /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 gtgtgctagc attagttatt
      301 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
      361 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      421 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      481 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
      541 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
      601 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
      661 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
      721 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
      781 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
      841 aggtctatat aagcagagct ggtttagtga acctgcaggt cggccgccac gaccggtgcc
      901 accatgactg aatacaaacc aactgttcgc ctggcaactc gtgatgatgt tccacgtgca
      961 gttcgcaccc tggctgctgc atttgctgac taccctgcaa cccgtcacac tgtggaccca
     1021 gaccgccaca ttgaacgtgt gactgaactg caggagctgt tcctgacccg tgtgggcctg
     1081 gacattggca aagtgtgggt ggcagatgat ggtgctgctg tggcagtgtg gaccacccct
     1141 gaatctgttg aagctggtgc agtgtttgct gagattggcc cacgcatggc agaactgtct
     1201 ggcagccgcc tggcagcaca acagcagatg gaaggtctgc tggcaccaca ccgcccaaaa
     1261 gaacctgctt ggttcctggc aactgtgggt gtgagccctg accaccaggg taagggcctg
     1321 ggctctgcag tggtgctgcc tggtgtggaa gcagctgaac gtgcaggtgt gcctgctttc
     1381 ctggagacct cagctccacg caacctgcct ttctatgaac gcctgggctt cactgtgact
     1441 gctgatgtgg aagtgccaga aggcccacgc acttggtgca tgactcgcaa accaggtgct
     1501 aggtgaccgc aataaaaaga cagaataaaa cgcacgggtg ttgggtcgtt tgttcactcg
     1561 gcccagagcc agcgaagtct gctcccgccc cgaaaaaggg ctccaagaag gcggtgacta
     1621 aggcgcagaa gaaaggcggc aagaagcgca agcgcagccg caaggagagc tattccatct
     1681 atgtgtacaa ggttctgaag caggtccacc ctgacaccgg catttcgtcc aaggccatgg
     1741 gcatcatgaa ttcgtttgtg aacgacattt tcgagcgcat cgcaggtgag gcttcccgcc
     1801 tggcgcatta caacaagcgc tcgaccatca cctccaggga gatccagacg gccgtgcgcc
     1861 tgctgctgcc tggggagttg gccaagcacg ccgtgtccga gggtactaag gccatcacca
     1921 agtacaccag cgctaaggat ccaccggtcg ccaccatggt gagcaagggc gaggagctgt
     1981 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     2041 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     2101 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
     2161 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     2221 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     2281 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     2341 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     2401 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc
     2461 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
     2521 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga
     2581 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     2641 ggatcactct cggcatggac gagctgtaca agtaaagcgg ccgcgactcc agttcgtggc
     2701 cgtcgatcgt ttaaagggag gtagtgagtc gagatatcga tactagtgat ccagacatga
     2761 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta
     2821 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag
     2881 ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt
     2941 tttaaagcaa gtaaaacctc tacaaatgtg gtatggctga ttatgatcct ctagagggac
     3001 agcccccccc caaagccccc agggatgtaa ttacgtccct cccccgctag ggggcagcag
     3061 cgagccgccc ggggctccgc tccggtccgg cgctcccccc gcatccccga gccggcagcg
     3121 tgcggggaca gcccgggcac ggggaaggtg gcacgggatc gctttcctct gaacgcttct
     3181 cgctgctctt tgagcctgca gacacctggg gggatacggg gaaaaagcca taccaatgga
     3241 gctcacgggg acagcccccc cccaaagccc ccagggatgt aattacgtcc ctcccccgct
     3301 agggggcagc agcgagccgc ccggggctcc gctccggtcc ggcgctcccc ccgcatcccc
     3361 gagccggcag cgtgcgggga cagcccgggc acggggaagg tggcacggga tcgctttcct
     3421 ctgaacgctt ctcgctgctc tttgagcctg cagacacctg gggggatacg gggaaaaaga
     3481 attcgataaa agttttgtta ctttatagaa gaaattttga gtttttgttt tttttaataa
     3541 ataaataaac ataaataaat tgtttgttga atttattatt agtatgtaag tgtaaatata
     3601 ataaaactta atatctattc aaattaataa ataaacctcg atatacagac cgataaaaca
     3661 catgcgtcaa ttttacacat gattatcttt aacgtacgtc acaatatgat tatctttcta
     3721 gggttaatct agctgcgtgt tctgcagcgt gtcgagcatc ttcatctgct ccatcacgct
     3781 gtaaaacaca tttgcaccgc gagtctgccc gtcctccacg ggttcaaaaa cgtgaatgaa
     3841 cgaggcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     3901 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     3961 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg
     4021 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     4081 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     4141 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     4201 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     4261 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     4321 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     4381 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     4441 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     4501 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     4561 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     4621 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     4681 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     4741 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     4801 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     4861 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     4921 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     4981 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     5041 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     5101 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     5161 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     5221 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     5281 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     5341 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     5401 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     5461 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     5521 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     5581 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     5641 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     5701 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     5761 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     5821 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     5881 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     5941 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6001 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6061 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6121 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     6181 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     6241 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     6301 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     6361 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     6421 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     6481 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     6541 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     6601 acaggaaaca gctatgacca tgattacgcc aagcgcgccc gccgggtaac tcacggggta
     6661 tccatgtcca tttctgcggc atccagccag gatacccgtc ctcgctgacg taatatccca
     6721 gcgccgcacc gctgtcatta atctgcacac cggcacggca gttccggctg tcgccggtat
     6781 tgttcgggtt gctgatgcgc ttcgggctga ccatccggaa ctgtgtccgg aaaagccgcg
     6841 acgaactggt atcccaggtg gcctgaacga acagttcacc gttaaaggcg tgcatggcca
     6901 caccttcccg aatcatcatg gtaaacgtgc gttttcgctc aacgtcaatg cagcagcagt
     6961 catcctcggc aaactctttc catgccgctt caacctcgcg ggaaaaggca cgggcttctt
     7021 cctccccgat gcccagatag cgccagcttg ggcgatgact gagccggaaa aaagacccga
     7081 cgatatgatc ctgatgcagc tagattaacc ctagaaagat agtctgcgta aaattgacgc
     7141 atg
//
