LOCUS       sequence-272363-        5490 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used as a donor vector containing N-term 3xFLAG to clone into pSLIK.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5490)
  AUTHORS   Pereira EJ, Burns JS, Lee CY, Marohl T, Calderon D, Wang L, Atkins 
            KA, Wang CC, Janes KA
  TITLE     Sporadic activation of an oxidative stress-dependent NRF2-p53 
            signaling network in breast epithelial spheroids and 
            premalignancies.
  JOURNAL   Sci Signal. 2020 Apr 14;13(627). pii: 13/627/eaba4200. doi: 
            10.1126/scisignal.aba4200.
  PUBMED    32291314
REFERENCE   2  (bases 1 to 5490)
  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: "Sci 
            Signal."; date: "2020-04-14"; volume: "13(627). pii: 
            13/627/eaba4200. doi"; pages: " 10.1126/scisignal.aba4200"
COMMENT     Sequence Label: pEN_TTmiRc2_3xflag_CHEK2
FEATURES             Location/Qualifiers
     source          1..5490
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     3..27
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             39..104
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    complement(1778..1877)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1989..2008)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2067..2086)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(2660..3193)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3382..3387,3388..3404,3405..3410))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     rep_origin      4067..4655
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4556..4575
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      4820..4906
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4998..5025
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     5033..5056
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    5075..5174
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(5206..5490,1..30)
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    5214..5232
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5250..5268
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5285..5303
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5321..5339
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5357..5375
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5392..5410
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5428..5446
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 agagctcgtt tagtgaaccg tcagatcgcc actagatgga ctacaaagac catgacggtg
       61 attataaaga tcatgacatc gattacaagg atgacgatga caagactaga atgtctcggg
      121 agtcggatgt tgaggctcag cagtctcatg gcagcagtgc ctgttcacag ccccatggca
      181 gcgttaccca gtcccaaggc tcctcctcac agtcccaggg catatccagc tcctctacca
      241 gcacgatgcc aaactccagc cagtcctctc actccagctc tgggacactg agctccttag
      301 agacagtgtc cactcaggaa ctctattcta ttcctgagga ccaagaacct gaggaccaag
      361 aacctgagga gcctacccct gccccctggg ctcgattatg ggcccttcag gatggatttg
      421 ccaatcttga atgtgtgaat gacaactact ggtttgggag ggacaaaagc tgtgaatatt
      481 gctttgatga accactgctg aaaagaacag ataaataccg aacatacagc aagaaacact
      541 ttcggatttt cagggaagtg ggtcctaaaa actcttacat tgcatacata gaagatcaca
      601 gtggcaatgg aacctttgta aatacagagc ttgtagggaa aggaaaacgc cgtcctttga
      661 ataacaattc tgaaattgca ctgtcactaa gcagaaataa agtttttgtc ttttttgatc
      721 tgactgtaga tgatcagtca gtttatccta aggcattaag agatgaatac atcatgtcaa
      781 aaactcttgg aagtggtgcc tgtggagagg taaagctggc tttcgagagg aaaacatgta
      841 agaaagtagc cataaagatc atcagcaaaa ggaagtttgc tattggttca gcaagagagg
      901 cagacccagc tctcaatgtt gaaacagaaa tagaaatttt gaaaaagcta aatcatcctt
      961 gcatcatcaa gattaaaaac ttttttgatg cagaagatta ttatattgtt ttggaattga
     1021 tggaaggggg agagctgttt gacaaagtgg tggggaataa acgcctgaaa gaagctacct
     1081 gcaagctcta tttttaccag atgctcttgg ctgtgcagta ccttcatgaa aacggtatta
     1141 tacaccgtga cttaaagcca gagaatgttt tactgtcatc tcaagaagag gactgtctta
     1201 taaagattac tgattttggg cactccaaga ttttgggaga gacctctctc atgagaacct
     1261 tatgtggaac ccccacctac ttggcgcctg aagttcttgt ttctgttggg actgctgggt
     1321 ataaccgtgc tgtggactgc tggagtttag gagttattct ttttatctgc cttagtgggt
     1381 atccaccttt ctctgagcat aggactcaag tgtcactgaa ggatcagatc accagtggaa
     1441 aatacaactt cattcctgaa gtctgggcag aagtctcaga gaaagctctg gaccttgtca
     1501 agaagttgtt ggtagtggat ccaaaggcac gttttacgac agaagaagcc ttaagacacc
     1561 cgtggcttca ggatgaagac atgaagagaa agtttcaaga tcttctgtct gaggaaaatg
     1621 aatccacagc tctaccccag gttctagccc agccttctac tagtcgaaag cggccccgtg
     1681 aaggggaagc cgagggtgcc gagaccacaa agcgcccagc tgtgtgtgct gctgtgttgt
     1741 gacaattgga agactaatgc gttttcgaga tatctagacc cagctttctt gtacaaagtt
     1801 ggcattataa gaaagcattg cttatcaatt tgttgcaacg aacaggtcac tatcagtcaa
     1861 aataaaatca ttatttgcca tccagctgca gctctggccc gtgtctcaaa atctctgatg
     1921 ttacattgca caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa
     1981 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa
     2041 ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc
     2101 aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca
     2161 tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac
     2221 ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt
     2281 actcaccact gcgatccccg gaaaaacagc attccaggta ttagaagaat atcctgattc
     2341 aggtgaaaat attgttgatg cgctggcagt gtccctgcgc cggttgcatt cgattcctgt
     2401 ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat
     2461 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga
     2521 acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca
     2581 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaggcc gggaagccga
     2641 tctcggcttg aacgaattgt taggtggcgg tacttgggtc gatatcaaag tgcatcactt
     2701 cttcccgtat gcccaacttt gtatagagag ccactgcggg atcgtcaccg taatctgctt
     2761 gcacgtagat cacataagca ccaagcgcgt tggcctcatg cttgaggaga ttgatgagcg
     2821 cggtggcaat gccctgcctc cggtgctcgc cggagactgc gagatcatag atatagatct
     2881 cactacgcgg ctgctcaaac ttgggcagaa cgtaagccgc gagagcgcca acaaccgctt
     2941 cttggtcgaa ggcagcaagc gcgatgaatg tcttactacg gagcaagttc ccgaggtaat
     3001 cggagtccgg ctgatgttgg gagtaggtgg ctacgtctcc gaactcacga ccgaaaagat
     3061 caagagcagc ccgcatggat ttgacttggt cagggccgag cctacatgtg cgaatgatgc
     3121 ccatacttga gccacctaac tttgttttag ggcgactgcc ctgctgcgta acatcgttgc
     3181 tgctgcgtaa catcgttgct gctccataac atcaaacatc gacccacggc gtaacgcgct
     3241 tgctgcttgg atgcccgagg catagactgt acaaaaaaac agtcataaca agccatgaaa
     3301 accgccactg cgccgttacc accgctgcgt tcggtcaagg ttctggacca gttgcgtgag
     3361 cgcatacgct acttgcatta cagtttacga accgaacagg cttatgtcaa ctgggttcgt
     3421 gccttcatcc gtttccacgg tgtgcgtcac ccggcaacct tgggcagcag cgaagtcgag
     3481 gcatttctgt cctggctggc gaacgagcgc aaggtttcgg tctccacgca tcgtcaggca
     3541 ttggcggcct tgctgttctt ctacggcaag gtgctgtgca cggatctgcc ctggcttcag
     3601 gagatcggaa gacctcggcc gtcgcggcgc ttgccggtgg tgctgacccc ggatgaagtg
     3661 gttcgcatcc tcggttttct ggaaggcgag catcgtttgt tcgcccagga ctctagctat
     3721 agttctagtg gttggctact aataggttgt attgatgttg gacgagtcgg aatcgcagac
     3781 cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag
     3841 aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat
     3901 ttgatgctcg atgagttttt ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc
     3961 attacgctga cttgacggga cggcgcaagc tcatgaccaa aatcccttaa cgtgagtttt
     4021 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4081 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4141 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4201 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4261 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4321 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4381 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4441 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4501 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4561 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4621 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4681 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     4741 ctgtggataa ccgtattacc gctagcatgg atctcgggga cgtctaacta ctaagcgaga
     4801 gtagggaact gccaggcatc aaataaaacg aaaggctcag tcggaagact gggcctttcg
     4861 ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga
     4921 tttgaacgtt gtgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc
     4981 caggcatcaa actaagcaga aggccatcct gacggatggc ctttttgcgt ttctacaaac
     5041 tcttcctgtt agttagttac ttaagctcgg gccccaaata atgattttat tttgactgat
     5101 agtgacctgt tcgttgcaac aaattgataa gcaatgcttt tttataatgc caactttgta
     5161 caaaaaagca ggctttaaag gaaccaattc agtcgactgg atcccgagtt tactccctat
     5221 cagtgataga gaacgtatgt cgagtttact ccctatcagt gatagagaac gatgtcgagt
     5281 ttactcccta tcagtgatag agaacgtatg tcgagtttac tccctatcag tgatagagaa
     5341 cgtatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt atccctatca
     5401 gtgatagaga acgtatgtcg agtttactcc ctatcagtga tagagaacgt atgtcgaggt
     5461 aggcgtgtac ggtgggaggc ctatataagc
//
