LOCUS       sequence-256393-        4819 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Piggybac vector for SRIRACCHA-mediated mutation enrichment (donor 
            repair template for HDR).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4819)
  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 4819)
  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: pBS-PtH2B
FEATURES             Location/Qualifiers
     source          1..4819
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    13..34
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(49..54,55..72,73..79)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    87..103
                     /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     92..114
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     111..127
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     111..127
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     146..166
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        148..166
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     195..211
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     196..212
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             870..923
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             930..1304
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="PEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVL
                     KQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPG
                     ELAKHAVSEGTKAITKYTSAK"
     regulatory      1317..1326
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1323..1325,1326..1328,1329..2042)
                     /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(1323..1325,1326..1328,1329..2039)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1368..1389)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1629..1648)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1976..1997
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2152..2171)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2153..2171)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2181..2198)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2181..2197)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2190..2212)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2338..2793)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2475..2496)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2687..2706
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2820..2924
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2925..2993,2994..3785)
                     /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(3143..3162)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3956..4544
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4445..4464
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4698..4715
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
       61 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
      121 ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctggg
      181 taccgggccc cccctcgagg tcgacggtat cgataagctt gcatgcctgc aggtcggccg
      241 ccacgaccgg tgccaccatg actgaataca aaccaactgt tcgcctggca actcgtgatg
      301 atgttccacg tgcagttcgc accctggctg ctgcatttgc tgactaccct gcaacccgtc
      361 acactgtgga cccagaccgc cacattgaac gtgtgactga actgcaggag ctgttcctga
      421 cccgtgtggg cctggacatt ggcaaagtgt gggtggcaga tgatggtgct gctgtggcag
      481 tgtggaccac ccctgaatct gttgaagctg gtgcagtgtt tgctgagatt ggcccacgca
      541 tggcagaact gtctggcagc cgcctggcag cacaacagca gatggaaggt ctgctggcac
      601 cacaccgccc aaaagaacct gcttggttcc tggcaactgt gggtgtgagc cctgaccacc
      661 agggtaaggg cctgggctct gcagtggtgc tgcctggtgt ggaagcagct gaacgtgcag
      721 gtgtgcctgc tttcctggag acctcagctc cacgcaacct gcctttctat gaacgcctgg
      781 gcttcactgt gactgctgat gtggaagtgc cagaaggccc acgcacttgg tgcatgactc
      841 gcaaaccagg tgctagcctt ggctccggcg agggcagggg aagtcttcta acatgcgggg
      901 acgtggagga aaatcccggc ccactcggcc cagagccagc gaagtctgct cccgccccga
      961 aaaagggctc caagaaggcg gtgactaagg cgcagaagaa aggcggcaag aagcgcaagc
     1021 gcagccgcaa ggagagctat tccatctatg tgtacaaggt tctgaagcag gtccaccctg
     1081 acaccggcat ttcgtccaag gccatgggca tcatgaattc gtttgtgaac gacattttcg
     1141 agcgcatcgc aggtgaggct tcccgcctgg cgcattacaa caagcgctcg accatcacct
     1201 ccagggagat ccagacggcc gtgcgcctgc tgctgcctgg ggagttggcc aagcacgccg
     1261 tgtccgaggg tactaaggcc atcaccaagt acaccagcgc taaggatcca ccggtcgcca
     1321 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     1381 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     1441 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     1501 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     1561 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     1621 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     1681 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     1741 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     1801 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     1861 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     1921 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     1981 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     2041 aaagcggccg cgactccagt tcgtggccgt cgatcgttta aagggaggta gtgagtcgag
     2101 atatcgatac tagttctaga gcggccgcca ccgcggtgga gctccaattc gccctatagt
     2161 gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     2221 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     2281 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggaaa
     2341 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     2401 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     2461 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     2521 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     2581 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     2641 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     2701 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     2761 ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc
     2821 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     2881 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     2941 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     3001 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     3061 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     3121 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     3181 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     3241 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     3301 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     3361 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     3421 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     3481 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     3541 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     3601 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     3661 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     3721 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     3781 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     3841 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     3901 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3961 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4021 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4081 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     4141 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4201 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4261 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4321 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4381 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4441 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4501 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4561 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     4621 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     4681 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     4741 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     4801 actggaaagc gggcagtga
//
