LOCUS       sequence-174398-        4989 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 4989)
  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 4989)
  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-MKK4-EE-NLS
FEATURES             Location/Qualifiers
     source          1..4989
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      19..607
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     508..527
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      772..858
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      950..977
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     985..1008
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    1027..1126
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1158..1472
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    1166..1184
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1202..1220
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1237..1255
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1273..1291
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1309..1327
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1344..1362
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1380..1398
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1445..1469
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1481..1546
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2660..2680
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(2719..2818)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2930..2949)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(3008..3027)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(3601..4134)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(4323..4328,4329..4345,4346..4351))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
ORIGIN
        1 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
       61 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
      121 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
      181 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      241 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      301 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      361 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      421 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      481 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      541 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      601 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      661 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgctagcat
      721 ggatctcggg gacgtctaac tactaagcga gagtagggaa ctgccaggca tcaaataaaa
      781 cgaaaggctc agtcggaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
      841 ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgtgaagca acggcccgga
      901 gggtggcggg caggacgccc gccataaact gccaggcatc aaactaagca gaaggccatc
      961 ctgacggatg gcctttttgc gtttctacaa actcttcctg ttagttagtt acttaagctc
     1021 gggccccaaa taatgatttt attttgactg atagtgacct gttcgttgca acaaattgat
     1081 aagcaatgct tttttataat gccaactttg tacaaaaaag caggctttaa aggaaccaat
     1141 tcagtcgact ggatcccgag tttactccct atcagtgata gagaacgtat gtcgagttta
     1201 ctccctatca gtgatagaga acgatgtcga gtttactccc tatcagtgat agagaacgta
     1261 tgtcgagttt actccctatc agtgatagag aacgtatgtc gagtttactc cctatcagtg
     1321 atagagaacg tatgtcgagt ttatccctat cagtgataga gaacgtatgt cgagtttact
     1381 ccctatcagt gatagagaac gtatgtcgag gtaggcgtgt acggtgggag gcctatataa
     1441 gcagagctcg tttagtgaac cgtcagatcg ccactagatg gactacaaag accatgacgg
     1501 tgattataaa gatcatgaca tcgattacaa ggatgacgat gacaagacta gtggatccat
     1561 gcagggtaaa cgcaaagcac tgaagttgaa ttttgcaaat ccacctttca aatctacagc
     1621 aaggtttact ctgaatccca atcctacagg agttcaaaac ccacacatag agagactgag
     1681 aacacacagc attgagtcat caggaaaact gaagatctcc cctgaacaac actgggattt
     1741 cactgcagag gacttgaaag accttggaga aattggacga ggagcttatg gttctgtcaa
     1801 caaaatggtc cacaaaccaa gtgggcaaat aatggcagtt aaaagaattc ggtcaacagt
     1861 ggatgaaaaa gaacaaaaac aacttcttat ggatttggat gtagtaatgc ggagtagtga
     1921 ttgcccatac attgttcagt tttatggtgc actcttcaga gagggtgact gttggatctg
     1981 tatggaactc atgtctacct cgtttgataa gttttacaaa tatgtatata gtgtattaga
     2041 tgatgttatt ccagaagaaa ttttaggcaa aatcacttta gcaactgtga aagcactaaa
     2101 ccacttaaaa gaaaacttga aaattattca cagagatatc aaaccttcca atattcttct
     2161 ggacagaagt ggaaatatca agctctgtga cttcggcatc agtggacagc ttgtagacga
     2221 gattgccaag gaaagagatg ctggctgtag gccatacatg gcacctgaaa gaatagaccc
     2281 aagcgcatca cgacaaggat atgatgtccg ctctgatgtc tggagtttgg ggatcacatt
     2341 gtatgagttg gccacaggcc gatttcctta tccaaagtgg aatagtgtat ttgatcaact
     2401 aacacaagtc gtgaaaggag atcctccgca gctgagtaat tctgaggaaa gggaattctc
     2461 cccgagtttc atcaactttg tcaacttgtg ccttacgaag gatgaatcca aaaggccaaa
     2521 gtataaagag cttctgaaac atccctttat tttgatgtat gaagaacgtg ccgttgaggt
     2581 cgcatgctat gtttgtaaaa tcctggatca aatgccagct actcccagct ctcccatgta
     2641 tgtcgatggt ggttctggtc cgaagaaaaa acgtaaagtg tagcaattgg aagactaatg
     2701 cgttttcgag atatctagac ccagctttct tgtacaaagt tggcattata agaaagcatt
     2761 gcttatcaat ttgttgcaac gaacaggtca ctatcagtca aaataaaatc attatttgcc
     2821 atccagctgc agctctggcc cgtgtctcaa aatctctgat gttacattgc acaagataaa
     2881 aatatatcat catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt
     2941 atgagccata ttcaacggga aacgtcgagg ccgcgattaa attccaacat ggatgctgat
     3001 ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcgc
     3061 ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc
     3121 aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg
     3181 accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc
     3241 ggaaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat
     3301 gcgctggcag tgtccctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac
     3361 agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat
     3421 gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg
     3481 cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat
     3541 aaccttattt ttgacgaggg gaaattaggc cgggaagccg atctcggctt gaacgaattg
     3601 ttaggtggcg gtacttgggt cgatatcaaa gtgcatcact tcttcccgta tgcccaactt
     3661 tgtatagaga gccactgcgg gatcgtcacc gtaatctgct tgcacgtaga tcacataagc
     3721 accaagcgcg ttggcctcat gcttgaggag attgatgagc gcggtggcaa tgccctgcct
     3781 ccggtgctcg ccggagactg cgagatcata gatatagatc tcactacgcg gctgctcaaa
     3841 cttgggcaga acgtaagccg cgagagcgcc aacaaccgct tcttggtcga aggcagcaag
     3901 cgcgatgaat gtcttactac ggagcaagtt cccgaggtaa tcggagtccg gctgatgttg
     3961 ggagtaggtg gctacgtctc cgaactcacg accgaaaaga tcaagagcag cccgcatgga
     4021 tttgacttgg tcagggccga gcctacatgt gcgaatgatg cccatacttg agccacctaa
     4081 ctttgtttta gggcgactgc cctgctgcgt aacatcgttg ctgctgcgta acatcgttgc
     4141 tgctccataa catcaaacat cgacccacgg cgtaacgcgc ttgctgcttg gatgcccgag
     4201 gcatagactg tacaaaaaaa cagtcataac aagccatgaa aaccgccact gcgccgttac
     4261 caccgctgcg ttcggtcaag gttctggacc agttgcgtga gcgcatacgc tacttgcatt
     4321 acagtttacg aaccgaacag gcttatgtca actgggttcg tgccttcatc cgtttccacg
     4381 gtgtgcgtca cccggcaacc ttgggcagca gcgaagtcga ggcatttctg tcctggctgg
     4441 cgaacgagcg caaggtttcg gtctccacgc atcgtcaggc attggcggcc ttgctgttct
     4501 tctacggcaa ggtgctgtgc acggatctgc cctggcttca ggagatcgga agacctcggc
     4561 cgtcgcggcg cttgccggtg gtgctgaccc cggatgaagt ggttcgcatc ctcggttttc
     4621 tggaaggcga gcatcgtttg ttcgcccagg actctagcta tagttctagt ggttggctac
     4681 taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca
     4741 tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat
     4801 atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt
     4861 tctaatcaga attggttaat tggttgtaac actggcagag cattacgctg acttgacggg
     4921 acggcgcaag ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     4981 ccccgtaga
//
