LOCUS       sequence-174433-        4938 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used as a donor vector containing N-term 3xFLAG to clone into 
            pSLIK-Hygro.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4938)
  AUTHORS   Shah M, Smolko CM, Kinicki S, Chapman ZD, Brautigan DL, Janes KA
  TITLE     Profiling Subcellular Protein Phosphatase Responses to 
            Coxsackievirus B3 Infection of Cardiomyocytes.
  JOURNAL   Mol Cell Proteomics. 2017 Apr;16(4 suppl 1):S244-S262. doi: 
            10.1074/mcp.O116.063487. Epub 2017 Feb 7.
  PUBMED    28174228
REFERENCE   2  (bases 1 to 4938)
  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: "Mol Cell 
            Proteomics."; date: "2017-04"; volume: "16(4 suppl 1):S244-S262. 
            doi"; pages: " 10.1074/mcp.O116.063487. Epub 2017 Feb 7"
COMMENT     Sequence Label: pEN_TTmiRc2 3XFLAG-MKK7-EE-NLS
FEATURES             Location/Qualifiers
     source          1..4938
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      18..606
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     507..526
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      771..857
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      949..976
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     984..1007
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    1026..1125
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1157..1471
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    1165..1183
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1201..1219
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1236..1254
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1272..1290
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1308..1326
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1343..1361
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1379..1397
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1444..1468
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1480..1545
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2608..2628
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(2667..2766)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2878..2897)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2956..2975)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(3549..4082)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(4271..4276,4277..4293,4294..4299))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
ORIGIN
        1 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
       61 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
      121 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
      181 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
      241 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
      301 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
      361 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
      421 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
      481 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
      541 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
      601 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
      661 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgctagcatg
      721 gatctcgggg acgtctaact actaagcgag agtagggaac tgccaggcat caaataaaac
      781 gaaaggctca gtcggaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc
      841 tcctgagtag gacaaatccg ccgggagcgg atttgaacgt tgtgaagcaa cggcccggag
      901 ggtggcgggc aggacgcccg ccataaactg ccaggcatca aactaagcag aaggccatcc
      961 tgacggatgg cctttttgcg tttctacaaa ctcttcctgt tagttagtta cttaagctcg
     1021 ggccccaaat aatgatttta ttttgactga tagtgacctg ttcgttgcaa caaattgata
     1081 agcaatgctt ttttataatg ccaactttgt acaaaaaagc aggctttaaa ggaaccaatt
     1141 cagtcgactg gatcccgagt ttactcccta tcagtgatag agaacgtatg tcgagtttac
     1201 tccctatcag tgatagagaa cgatgtcgag tttactccct atcagtgata gagaacgtat
     1261 gtcgagttta ctccctatca gtgatagaga acgtatgtcg agtttactcc ctatcagtga
     1321 tagagaacgt atgtcgagtt tatccctatc agtgatagag aacgtatgtc gagtttactc
     1381 cctatcagtg atagagaacg tatgtcgagg taggcgtgta cggtgggagg cctatataag
     1441 cagagctcgt ttagtgaacc gtcagatcgc cactagatgg actacaaaga ccatgacggt
     1501 gattataaag atcatgacat cgattacaag gatgacgatg acaagactag aggatccatg
     1561 ctggggctcc catcaacctt gttcacaccg cgcagtatgg agagcatcga gattgaccag
     1621 aagctgcagg agatcatgaa gcagacaggg tacctgacta tcgggggcca gcgttatcag
     1681 gcagaaatca atgacttgga gaacttgggt gagatgggca gtggtacctg tggtcaggtg
     1741 tggaagatgc ggttccggaa gacaggccac atcattgctg ttaagcaaat gcggcgctct
     1801 gggaacaagg aagagaataa gcgcattttg atggacctgg atgtagtact caagagccat
     1861 gactgccctt acatcgttca gtgctttggc accttcatca ccaacacaga cgtctttatt
     1921 gccatggagc tcatgggcac atgtgcagag aagctgaaga aacgaatgca gggccccatt
     1981 ccagagcgaa tcctgggcaa gatgactgtg gcgattgtga aagcactgta ctatctgaag
     2041 gagaagcatg gcgtcatcca tcgcgatgtc aaaccctcca acatcctgct agatgagcgg
     2101 ggccagatca agctctgtga ctttggcatc agtggccgcc ttgttgacga aaaagccaaa
     2161 gaacggagtg ctggctgtgc tgcctatatg gctcccgagc gcatcgaccc tccagatccc
     2221 accaagcctg actatgacat ccgagctgat gtgtggagcc tgggcatctc actggtggag
     2281 ctggcaacag gacagttccc ctataagaac tgcaagacgg actttgaggt cctcaccaaa
     2341 gtcctacagg aagagccccc actcctgcct ggtcacatgg gcttctcagg ggacttccag
     2401 tcatttgtca aagactgcct tactaaagat cacaggaaga gaccaaagta taataagcta
     2461 cttgaacaca gcttcatcaa gcactatgag atactcgagg tggatgtcgc gtcctggttt
     2521 aaggatgtca tggcgaagac cgagtcccca aggactagtg gagtcctgag tcagcaccat
     2581 ctgcccttct tcaggggtgg ttctggtccg aagaaaaaac gtaaagtgta gcaattggaa
     2641 gactaatgcg ttttcgagat atctagaccc agctttcttg tacaaagttg gcattataag
     2701 aaagcattgc ttatcaattt gttgcaacga acaggtcact atcagtcaaa ataaaatcat
     2761 tatttgccat ccagctgcag ctctggcccg tgtctcaaaa tctctgatgt tacattgcac
     2821 aagataaaaa tatatcatca tgaacaataa aactgtctgc ttacataaac agtaatacaa
     2881 ggggtgttat gagccatatt caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg
     2941 atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa
     3001 tctatcgctt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta
     3061 gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc
     3121 ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg
     3181 cgatccccgg aaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata
     3241 ttgttgatgc gctggcagtg tccctgcgcc ggttgcattc gattcctgtt tgtaattgtc
     3301 cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt
     3361 tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga
     3421 aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct
     3481 cacttgataa ccttattttt gacgagggga aattaggccg ggaagccgat ctcggcttga
     3541 acgaattgtt aggtggcggt acttgggtcg atatcaaagt gcatcacttc ttcccgtatg
     3601 cccaactttg tatagagagc cactgcggga tcgtcaccgt aatctgcttg cacgtagatc
     3661 acataagcac caagcgcgtt ggcctcatgc ttgaggagat tgatgagcgc ggtggcaatg
     3721 ccctgcctcc ggtgctcgcc ggagactgcg agatcataga tatagatctc actacgcggc
     3781 tgctcaaact tgggcagaac gtaagccgcg agagcgccaa caaccgcttc ttggtcgaag
     3841 gcagcaagcg cgatgaatgt cttactacgg agcaagttcc cgaggtaatc ggagtccggc
     3901 tgatgttggg agtaggtggc tacgtctccg aactcacgac cgaaaagatc aagagcagcc
     3961 cgcatggatt tgacttggtc agggccgagc ctacatgtgc gaatgatgcc catacttgag
     4021 ccacctaact ttgttttagg gcgactgccc tgctgcgtaa catcgttgct gctgcgtaac
     4081 atcgttgctg ctccataaca tcaaacatcg acccacggcg taacgcgctt gctgcttgga
     4141 tgcccgaggc atagactgta caaaaaaaca gtcataacaa gccatgaaaa ccgccactgc
     4201 gccgttacca ccgctgcgtt cggtcaaggt tctggaccag ttgcgtgagc gcatacgcta
     4261 cttgcattac agtttacgaa ccgaacaggc ttatgtcaac tgggttcgtg ccttcatccg
     4321 tttccacggt gtgcgtcacc cggcaacctt gggcagcagc gaagtcgagg catttctgtc
     4381 ctggctggcg aacgagcgca aggtttcggt ctccacgcat cgtcaggcat tggcggcctt
     4441 gctgttcttc tacggcaagg tgctgtgcac ggatctgccc tggcttcagg agatcggaag
     4501 acctcggccg tcgcggcgct tgccggtggt gctgaccccg gatgaagtgg ttcgcatcct
     4561 cggttttctg gaaggcgagc atcgtttgtt cgcccaggac tctagctata gttctagtgg
     4621 ttggctacta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga
     4681 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt
     4741 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga
     4801 tgagtttttc taatcagaat tggttaattg gttgtaacac tggcagagca ttacgctgac
     4861 ttgacgggac ggcgcaagct catgaccaaa atcccttaac gtgagttttc gttccactga
     4921 gcgtcagacc ccgtagaa
//
