LOCUS       sequence-267560-        4729 bp    DNA     circular SYN 15-APR-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 4729)
  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 4729)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-PtH
FEATURES             Location/Qualifiers
     source          1..4729
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..18
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    135..156
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(171..176,177..194,195..201)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    209..225
                     /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     214..236
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     233..249
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     233..249
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     268..288
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        270..288
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     317..333
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     318..334
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             992..1045
                     /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             1052..2074
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             1052..2074
                     /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(2184..2203)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2185..2203)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2213..2230)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2213..2229)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2222..2244)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2370..2825)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2507..2528)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2719..2738
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2852..2956
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2957..3025,3026..3817)
                     /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(3175..3194)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3988..4576
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4477..4496
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
       61 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
      121 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
      181 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
      241 agctatgacc atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg
      301 ggtaccgggc cccccctcga ggtcgacggt atcgataagc ttgcatgcct gcaggtcggc
      361 cgccacgacc ggtgccacca tgactgaata caaaccaact gttcgcctgg caactcgtga
      421 tgatgttcca cgtgcagttc gcaccctggc tgctgcattt gctgactacc ctgcaacccg
      481 tcacactgtg gacccagacc gccacattga acgtgtgact gaactgcagg agctgttcct
      541 gacccgtgtg ggcctggaca ttggcaaagt gtgggtggca gatgatggtg ctgctgtggc
      601 agtgtggacc acccctgaat ctgttgaagc tggtgcagtg tttgctgaga ttggcccacg
      661 catggcagaa ctgtctggca gccgcctggc agcacaacag cagatggaag gtctgctggc
      721 accacaccgc ccaaaagaac ctgcttggtt cctggcaact gtgggtgtga gccctgacca
      781 ccagggtaag ggcctgggct ctgcagtggt gctgcctggt gtggaagcag ctgaacgtgc
      841 aggtgtgcct gctttcctgg agacctcagc tccacgcaac ctgcctttct atgaacgcct
      901 gggcttcact gtgactgctg atgtggaagt gccagaaggc ccacgcactt ggtgcatgac
      961 tcgcaaacca ggtgctagcc ttggctccgg cgagggcagg ggaagtcttc taacatgcgg
     1021 ggacgtggag gaaaatcccg gcccactcgg caaaaagcct gaactcaccg cgacgtctgt
     1081 cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg
     1141 cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa
     1201 tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc
     1261 gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat
     1321 ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt
     1381 tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag
     1441 cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat
     1501 atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag
     1561 tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt
     1621 ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat
     1681 aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa
     1741 catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg
     1801 gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct
     1861 tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg
     1921 tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg
     1981 cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg
     2041 acgccccagc actcgtccga gggcaaagga atagagcggc cgcgactcca gttcgtggcc
     2101 gtcgatcgtt taaagggagg tagtgagtcg agatatcgat actagttcta gagcggccgc
     2161 caccgcggtg gagctccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg
     2221 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     2281 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     2341 aacagttgcg cagcctgaat ggcgaatgga aattgtaagc gttaatattt tgttaaaatt
     2401 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     2461 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     2521 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     2581 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     2641 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     2701 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     2761 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     2821 cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     2881 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     2941 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     3001 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     3061 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     3121 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     3181 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     3241 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     3301 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     3361 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     3421 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     3481 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     3541 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     3601 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     3661 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     3721 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     3781 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     3841 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     3901 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     3961 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     4021 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4081 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     4141 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     4201 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     4261 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     4321 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     4381 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     4441 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     4501 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     4561 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     4621 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     4681 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgc
//
