LOCUS       sequence-174436-        4629 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 4629)
  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 4629)
  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-EGFP-NLS
FEATURES             Location/Qualifiers
     source          1..4629
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      14..602
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     503..522
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      767..853
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      945..972
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     980..1003
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    1022..1121
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1153..1467
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    1161..1179
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1197..1215
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1232..1250
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1268..1286
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1304..1322
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1339..1357
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1375..1393
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1440..1464
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1476..1541
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             join(1548..1550,1551..2261)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1590..1611)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1851..1870)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2198..2219
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2295..2315
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(2354..2453)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2565..2584)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2643..2662)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(3236..3769)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3958..3963,3964..3980,3981..3986))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
ORIGIN
        1 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
       61 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
      121 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
      181 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
      241 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
      301 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
      361 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
      421 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
      481 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
      541 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
      601 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
      661 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgct agcatggatc
      721 tcggggacgt ctaactacta agcgagagta gggaactgcc aggcatcaaa taaaacgaaa
      781 ggctcagtcg gaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct
      841 gagtaggaca aatccgccgg gagcggattt gaacgttgtg aagcaacggc ccggagggtg
      901 gcgggcagga cgcccgccat aaactgccag gcatcaaact aagcagaagg ccatcctgac
      961 ggatggcctt tttgcgtttc tacaaactct tcctgttagt tagttactta agctcgggcc
     1021 ccaaataatg attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgataagca
     1081 atgctttttt ataatgccaa ctttgtacaa aaaagcaggc tttaaaggaa ccaattcagt
     1141 cgactggatc ccgagtttac tccctatcag tgatagagaa cgtatgtcga gtttactccc
     1201 tatcagtgat agagaacgat gtcgagttta ctccctatca gtgatagaga acgtatgtcg
     1261 agtttactcc ctatcagtga tagagaacgt atgtcgagtt tactccctat cagtgataga
     1321 gaacgtatgt cgagtttatc cctatcagtg atagagaacg tatgtcgagt ttactcccta
     1381 tcagtgatag agaacgtatg tcgaggtagg cgtgtacggt gggaggccta tataagcaga
     1441 gctcgtttag tgaaccgtca gatcgccact agatggacta caaagaccat gacggtgatt
     1501 ataaagatca tgacatcgat tacaaggatg acgatgacaa gactagtgtg agcaagggcg
     1561 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     1621 acaagttcag cgtgtctggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
     1681 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
     1741 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
     1801 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
     1861 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     1921 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     1981 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggcgaact
     2041 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     2101 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt
     2161 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     2221 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtccaagggt gggcgcgccg
     2281 acggtggttc tggtccgaag aaaaaacgta aagtgtagca attggaagac taatgcgttt
     2341 tcgagatatc tagacccagc tttcttgtac aaagttggca ttataagaaa gcattgctta
     2401 tcaatttgtt gcaacgaaca ggtcactatc agtcaaaata aaatcattat ttgccatcca
     2461 gctgcagctc tggcccgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat
     2521 atcatcatga acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag
     2581 ccatattcaa cgggaaacgt cgaggccgcg attaaattcc aacatggatg ctgatttata
     2641 tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta
     2701 tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga
     2761 tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat
     2821 caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga tccccggaaa
     2881 aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct
     2941 ggcagtgtcc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga
     3001 tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag
     3061 tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa
     3121 acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct
     3181 tatttttgac gaggggaaat taggccggga agccgatctc ggcttgaacg aattgttagg
     3241 tggcggtact tgggtcgata tcaaagtgca tcacttcttc ccgtatgccc aactttgtat
     3301 agagagccac tgcgggatcg tcaccgtaat ctgcttgcac gtagatcaca taagcaccaa
     3361 gcgcgttggc ctcatgcttg aggagattga tgagcgcggt ggcaatgccc tgcctccggt
     3421 gctcgccgga gactgcgaga tcatagatat agatctcact acgcggctgc tcaaacttgg
     3481 gcagaacgta agccgcgaga gcgccaacaa ccgcttcttg gtcgaaggca gcaagcgcga
     3541 tgaatgtctt actacggagc aagttcccga ggtaatcgga gtccggctga tgttgggagt
     3601 aggtggctac gtctccgaac tcacgaccga aaagatcaag agcagcccgc atggatttga
     3661 cttggtcagg gccgagccta catgtgcgaa tgatgcccat acttgagcca cctaactttg
     3721 ttttagggcg actgccctgc tgcgtaacat cgttgctgct gcgtaacatc gttgctgctc
     3781 cataacatca aacatcgacc cacggcgtaa cgcgcttgct gcttggatgc ccgaggcata
     3841 gactgtacaa aaaaacagtc ataacaagcc atgaaaaccg ccactgcgcc gttaccaccg
     3901 ctgcgttcgg tcaaggttct ggaccagttg cgtgagcgca tacgctactt gcattacagt
     3961 ttacgaaccg aacaggctta tgtcaactgg gttcgtgcct tcatccgttt ccacggtgtg
     4021 cgtcacccgg caaccttggg cagcagcgaa gtcgaggcat ttctgtcctg gctggcgaac
     4081 gagcgcaagg tttcggtctc cacgcatcgt caggcattgg cggccttgct gttcttctac
     4141 ggcaaggtgc tgtgcacgga tctgccctgg cttcaggaga tcggaagacc tcggccgtcg
     4201 cggcgcttgc cggtggtgct gaccccggat gaagtggttc gcatcctcgg ttttctggaa
     4261 ggcgagcatc gtttgttcgc ccaggactct agctatagtt ctagtggttg gctactaata
     4321 ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta
     4381 tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt
     4441 attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa
     4501 tcagaattgg ttaattggtt gtaacactgg cagagcatta cgctgacttg acgggacggc
     4561 gcaagctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     4621 tagaaaaga
//
