LOCUS       sequence-267641-        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"
     primer_bind     68..85
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    202..223
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(238..243,244..261,262..268)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    276..292
                     /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     281..303
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     300..316
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     300..316
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     335..355
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        337..355
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     384..400
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     385..401
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1059..1112
                     /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             1119..1493
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="PEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVL
                     KQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPG
                     ELAKHAVSEGTKAITKYTSAK"
     regulatory      1506..1515
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1512..1514,1515..1517,1518..2231)
                     /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(1512..1514,1515..1517,1518..2228)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1557..1578)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1818..1837)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2165..2186
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2341..2360)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2342..2360)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2370..2387)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2370..2386)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2379..2401)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2527..2982)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2664..2685)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2876..2895
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3009..3113
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3114..3182,3183..3974)
                     /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(3332..3351)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4145..4733
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4634..4653
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
       61 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
      121 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
      181 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
      241 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
      301 aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca
      361 aaagctgggt accgggcccc ccctcgaggt cgacggtatc gataagcttg catgcctgca
      421 ggtcggccgc cacgaccggt gccaccatga ctgaatacaa accaactgtt cgcctggcaa
      481 ctcgtgatga tgttccacgt gcagttcgca ccctggctgc tgcatttgct gactaccctg
      541 caacccgtca cactgtggac ccagaccgcc acattgaacg tgtgactgaa ctgcaggagc
      601 tgttcctgac ccgtgtgggc ctggacattg gcaaagtgtg ggtggcagat gatggtgctg
      661 ctgtggcagt gtggaccacc cctgaatctg ttgaagctgg tgcagtgttt gctgagattg
      721 gcccacgcat ggcagaactg tctggcagcc gcctggcagc acaacagcag atggaaggtc
      781 tgctggcacc acaccgccca aaagaacctg cttggttcct ggcaactgtg ggtgtgagcc
      841 ctgaccacca gggtaagggc ctgggctctg cagtggtgct gcctggtgtg gaagcagctg
      901 aacgtgcagg tgtgcctgct ttcctggaga cctcagctcc acgcaacctg cctttctatg
      961 aacgcctggg cttcactgtg actgctgatg tggaagtgcc agaaggccca cgcacttggt
     1021 gcatgactcg caaaccaggt gctagccttg gctccggcga gggcagggga agtcttctaa
     1081 catgcgggga cgtggaggaa aatcccggcc cactcggccc agagccagcg aagtctgctc
     1141 ccgccccgaa aaagggctcc aagaaggcgg tgactaaggc gcagaagaaa ggcggcaaga
     1201 agcgcaagcg cagccgcaag gagagctatt ccatctatgt gtacaaggtt ctgaagcagg
     1261 tccaccctga caccggcatt tcgtccaagg ccatgggcat catgaattcg tttgtgaacg
     1321 acattttcga gcgcatcgca ggtgaggctt cccgcctggc gcattacaac aagcgctcga
     1381 ccatcacctc cagggagatc cagacggccg tgcgcctgct gctgcctggg gagttggcca
     1441 agcacgccgt gtccgagggt actaaggcca tcaccaagta caccagcgct aaggatccac
     1501 cggtcgccac catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     1561 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     1621 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     1681 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     1741 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     1801 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     1861 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     1921 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     1981 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     2041 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     2101 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     2161 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     2221 tgtacaagta aagcggccgc gactccagtt cgtggccgtc gatcgtttaa agggaggtag
     2281 tgagtcgaga tatcgatact agttctagag cggccgccac cgcggtggag ctccaattcg
     2341 ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg
     2401 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     2461 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     2521 gaatggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     2581 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     2641 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     2701 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     2761 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     2821 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     2881 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     2941 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg
     3001 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     3061 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     3121 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     3181 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     3241 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     3301 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     3361 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     3421 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     3481 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     3541 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     3601 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     3661 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     3721 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     3781 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     3841 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     3901 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     3961 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     4021 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     4081 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     4141 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     4201 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     4261 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     4321 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     4381 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     4441 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     4501 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     4561 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     4621 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     4681 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     4741 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     4801 ctgcgttatc ccctgattc
//
