LOCUS       sequence-66854-e        5530 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For constitutive or doxycycline-inducible expression of the site 
            specific homing nuclease I-PpoI (human codon optimized). Confers 
            resistance to puro. Use T-REx cells for doxy-inducible expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5530)
  AUTHORS   Britton S, Coates J, Jackson SP
  TITLE     A new method for high-resolution imaging of Ku foci to decipher 
            mechanisms of DNA double-strand break repair.
  JOURNAL   J Cell Biol. 2013 Jul 29.
  PUBMED    23897892
REFERENCE   2  (bases 1 to 5530)
  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"; journalName: "J Cell 
            Biol. 2013 Jul 29."
COMMENT     Sequence Label: pICE-HA-NLS-I-PpoI
FEATURES             Location/Qualifiers
     source          1..5530
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(40..57)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    46..270
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      316..744
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(403..422)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     613..634
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(753..773)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        758..1087
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      938..1073
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1000..1019
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1139..1738
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1139..1158)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1635..1655
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1868..1989
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1905..1924)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1959..1978
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2039..2055)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2039..2055)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2052..2074)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2063..2079
                     /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)."
     promoter        complement(join(2087..2093,2094..2111,2112..2117))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2132..2153
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2270..2287)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2441..3026)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2521..2540)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3197..3988,3989..4057))
                     /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     3820..3839
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4058..4162)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        4234..4613
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4614..4817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4768..4788
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    4819..4837
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4840..4858
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     4868..4892
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             4978..5004
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             5008..5028
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 tcgagacgcg tctgcagtct agataaaccc gctgatcagc ctcgactgtg ccttctagtt
       61 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
      121 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
      181 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
      241 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
      301 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
      361 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
      421 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
      481 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
      541 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
      601 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
      661 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
      721 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
      781 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
      841 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
      901 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
      961 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     1021 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     1081 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc acgtgaccat
     1141 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg
     1201 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg
     1261 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     1321 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag
     1381 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     1441 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     1501 cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag
     1561 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
     1621 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
     1681 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaca
     1741 cgtgctacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     1801 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     1861 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     1921 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     1981 tgtatcttat catgtctggc tagccgtcga cctctagcta gagcttggcg taatcatggt
     2041 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     2101 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     2161 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     2221 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     2281 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     2341 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     2401 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     2461 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     2521 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     2581 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     2641 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     2701 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     2761 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     2821 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     2881 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     2941 gctcttgatc cggcaaacaa accaccgctg gtagcggttt ttttgtttgc aagcagcaga
     3001 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     3061 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     3121 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     3181 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     3241 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     3301 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     3361 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     3421 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     3481 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     3541 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     3601 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     3661 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     3721 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     3781 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     3841 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     3901 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     3961 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     4021 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     4081 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     4141 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
     4201 cgggagatcc tgtacgggcc agatatccgc gttgacattg attattgact agttattaat
     4261 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
     4321 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
     4381 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
     4441 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
     4501 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
     4561 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
     4621 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
     4681 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
     4741 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
     4801 tctatataag cagagctctc cctatcagtg atagagatct ccctatcagt gatagagatc
     4861 gtcgacgagc tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg
     4921 acctccatag aagacaccgg gaccgatcca gcctccggca agcttgccgc caccatgtac
     4981 ccctacgacg tgcccgatta cgccggaccc aagaaaaagc ggaaagtggg cggatccatg
     5041 gccctgacca acgcccagat cctggccgtg atcgacagct gggaggaaac cgtgggccag
     5101 ttccccgtga tcacccacca tgtgcctctg ggcggaggcc tccagggaac cctgcactgt
     5161 tacgagatcc ccctggccgc tccttacggc gtgggctttg ccaagaacgg ccccaccaga
     5221 tggcagtaca agcggaccat caaccaggtg gtgcacagat ggggcagcca caccgtgccc
     5281 tttctgctgg aacccgacaa catcaacggc aagacctgca ccgccagcca cctgtgccac
     5341 aacaccagat gccacaaccc cctgcacctg tgctgggaga gcctggacga caacaagggc
     5401 cggaattggt gccctggccc taatggcgga tgtgtgcatg ccgtcgtgtg cctgagacag
     5461 ggacctctgt atggccctgg cgctacagtg gctggccctc agcagagggg ctcccacttc
     5521 gtggtgtaac
//
