LOCUS       sequence-256392-        4531 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 4531)
  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 4531)
  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-BtH
FEATURES             Location/Qualifiers
     source          1..4531
                     /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"
     regulatory      252..261
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             258..653
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             672..725
                     /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             732..1754
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             732..1754
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     primer_bind     complement(1864..1883)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1865..1883)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1893..1910)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1893..1909)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1902..1924)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2050..2505)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2187..2208)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2399..2418
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2532..2636
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2637..2705,2706..3497)
                     /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(2855..2874)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3668..4256
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4157..4176
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4410..4427
                     /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 gccaagcctt tgtctcaaga agaatccacc ctcattgaaa
      301 gagcaacggc tacaatcaac agcatcccca tctctgaaga ctacagcgtc gccagcgcag
      361 ctctctctag cgacggccgc atcttcactg gtgtcaatgt atatcatttt actgggggac
      421 cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc ggcagctggc aacctgactt
      481 gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag cccctgcgga cggtgccgac
      541 aggtgcttct cgatctgcat cctgggatca aagccatagt gaaggacagt gatggacagc
      601 cgacggcagt tgggattcgt gaattgctgc cctctggtta tgtgtgggag ggcgctagcc
      661 ttggctccgg cgagggcagg ggaagtcttc taacatgcgg ggacgtggag gaaaatcccg
      721 gcccactcgg caaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa
      781 agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca
      841 gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct
      901 acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc
      961 ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg
     1021 tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg
     1081 ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac
     1141 cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc
     1201 atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc
     1261 tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg
     1321 atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga
     1381 gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt
     1441 ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag
     1501 gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct
     1561 tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc
     1621 gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga
     1681 ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga
     1741 gggcaaagga atagagcggc cgcgactcca gttcgtggcc gtcgatcgtt taaagggagg
     1801 tagtgagtcg agatatcgat actagttcta gagcggccgc caccgcggtg gagctccaat
     1861 tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac
     1921 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     1981 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     2041 ggcgaatgga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     2101 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     2161 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     2221 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     2281 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     2341 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     2401 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     2461 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc
     2521 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     2581 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     2641 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     2701 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     2761 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     2821 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     2881 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     2941 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     3001 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     3061 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     3121 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     3181 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     3241 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     3301 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     3361 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     3421 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     3481 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     3541 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     3601 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     3661 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     3721 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     3781 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     3841 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     3901 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     3961 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     4021 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     4081 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     4141 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     4201 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     4261 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     4321 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     4381 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     4441 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     4501 acaggtttcc cgactggaaa gcgggcagtg a
//
