LOCUS       sequence-57041-e        8172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TimeSTAMP method for tagging and visualization of newly synthesized 
            proteins. TimeSTAMPa cassette used for for low abundance proteins or
            proteins with slow ongoing synthesis rates.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8172)
  AUTHORS   Lin MZ, Tsien RY
  TITLE     TimeSTAMP tagging of newly synthesized proteins.
  JOURNAL   Curr Protoc Protein Sci. 2010 Feb;Chapter 26:Unit 26.5. doi: 
            10.1002/0471140864.ps2605s59.
  PUBMED    20155731
REFERENCE   2  (bases 1 to 8172)
  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: "Curr Protoc
            Protein Sci."; date: "2010-02"; volume: "Chapter 26:Unit 26.5. doi";
            pages: " 10.1002/0471140864.ps2605s59"
COMMENT     Sequence Label: pCS6-mPSD95a-TSa-HA
FEATURES             Location/Qualifiers
     source          1..8172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     505..529
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2918..2941
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3638..3664
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(4573..4597)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(4598..4617)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4599..4617)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4628..4645)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4628..4644)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4637..4659)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     polyA_signal    5049..5183
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5086..5105)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5140..5159
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5309..5764
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5396..5415)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5606..5627
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             join(6052..6120,6121..6912)
                     /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     complement(6270..6289)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     protein_bind    7014..7047
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      7125..7713
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7614..7633
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7867..7884
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg
      541 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccggactct
      601 agcctaggcc gcggccaggc cccccaacat ggactgtctc tgtatagtga caaccaagaa
      661 ataccgctac caagatgaag acacgccccc tctggaacac agcccggccc acctccccaa
      721 ccaggccaat tctccccctg tgattgtcaa cacggacacc ctagaagccc caggatatgt
      781 gaacggaaca gagggggaga tggagtatga ggagatcaca ttggaaaggg gtaactcagg
      841 tctgggcttc agcatcgcag gtggcaccga caacccacac atcggtgacg acccatccat
      901 ctttatcacc aagatcattc ctggtggggc tgcagcccag gatggccgcc tcagggtcaa
      961 cgacagcatc ctgtttgtca atgaagtgga tgtccgggag gtgacccatt cagctgcagt
     1021 ggaggccctc aaagaggcgg gttccatcgt tcgcctctac gtcatgcgcc ggaaaccccc
     1081 agctgagaag atcatagaga tcaagcttat caaagggcct aaaggacttg gcttcagcat
     1141 cgcagggggc gttgggaacc agcacatccc tggagataat agcatctacg taaccaagat
     1201 catcgaagga ggcgctgccc acaaggaggg caggttgcag atcggagaca agatcctggc
     1261 ggtcaacagt gtggggctag aggatgtcat gcatgaggac gccgtggcag ccctgaagaa
     1321 cacatatgac gttgtgtacc taaaggtggc caagcccagc aatgcctacc tgagtgacag
     1381 ctatgctccc ccagacatca caacctcata ttctcagcac ctggacaatg agatcagtca
     1441 tagcagctac ttgggcaccg actaccccac agccatgacc cccacttccc ctcggcgcta
     1501 ctcccccgtg gccaaggacc tgctagggga ggaagatatt ccccgggaac caaggcggat
     1561 cgtgatccat cggggctcca ccggcctggg cttcaacatt gtgggcggcg aggacggtga
     1621 aggcatcttc atctccttca tccttgctgg gggtccagct gacctcagtg gggagctacg
     1681 gaagggggac cagatcctgt cggtcaatgg tgttgacctc cgcaatgcca gtcatgaaca
     1741 ggctgccatt gccctgaaga acgcgggtca gacggtcacg atcatcgctc agtataaacc
     1801 agaagagtat agccgattcg aggccaagat ccatgatctt cgggaacagc ttatgaatag
     1861 tagcctgggc tcagggactg catctctgcg aagcaacccc aagcggggct tctatatcag
     1921 ggccctgttt gactacgaca agaccaagga ctgcggtttc ttgagccagg ccctgagctt
     1981 ccactttggg gatgtgcttc atgtaattga cgccagcgac gaagagtggt ggcaagcgcg
     2041 gcgggtccac tctgacagtg agaccgatga cattggcttc attcccagca aacggcgggt
     2101 cgagcgacga gagtggtcaa ggttaaaggc caaggactgg ggctccagct ctggatcaca
     2161 gggtcgagaa gactcggttc tgagctatga gacggtgacg cagatggaag tgcactacgc
     2221 tcgccccatc atcatccttg ggcctaccaa agaccgtgcc aacgatgatc ttctctccga
     2281 gttccccgac aagtttggat cctgtgtccc tcatacgaca cgtcctaagc gggaatatga
     2341 gatagacggc cgcgattacc actttgtctc ctcccgggag aaaatggaga aggacattca
     2401 ggcgcacaag ttcattgagg ctggccagta caacagccac ctctacggga ccagcgtcca
     2461 gtctgtgcga gaggtagcag agcaggggaa gcactgcatc cttgatgtct cagccaatgc
     2521 cgtgcggcgg ctgcaggcgg cccacctgca ccctatcgcc atcttcatcc gtccccgctc
     2581 cctggagaat gtgctagaga tcaataagcg gatcacagag gagcaagccc ggaaagcctt
     2641 cgacagggcc acgaagctgg agcaggagtt cacggagtgc ttctcagcca tcgtagaggg
     2701 cgacagcttt gaagagatct atcacaaagt gaaacgtgtc atcgaagacc tctcaggccc
     2761 ctacatctgg gtcccagccc gagagagact ggagcccggg cccatggcca gcatgactgg
     2821 tggacagcaa atggggtcga cgctctacca ggagttcgat gagatggaag agtgctctca
     2881 gcacttaccc ggcgccggca gtagtggcga tatcatggat tacaaggatg acgacgataa
     2941 gggctcttcc gggacaggct ccggatccgg cactagtgcg cccatcacgg cgtacgccca
     3001 gcagacgaga ggcctcctag ggtgtataat caccagcctg actggccggg acaaaaacca
     3061 agtggagggt gaggtccaga tcgtgtcaac tgctacccaa accttcctgg caacgtgcat
     3121 caatggggta tgctgggcag tctaccacgg ggccggaacg aggaccatcg catcacccaa
     3181 gggtcctgtc atccagatgt ataccaatgt ggaccaagac cttgtgggct ggcccgctcc
     3241 tcaaggttcc cgctcattga caccctgtac ctgcggctcc tcggaccttt acctggtcac
     3301 gaggcacgcc gatgtcattc ccgtgcgccg gcgaggtgat agcaggggta gcctgctttc
     3361 gccccggccc atttcctact tgaaaggctc ctcggggggt ccgctgttgt gccccgcggg
     3421 acacgccgtg ggcctattca gggccgcggt gtgcacccgt ggagtggcta aagcggtgga
     3481 ctttatccct gtggagaacc tagagacaac catgagatcc ccggtgttca cggacaactc
     3541 ctctccacca gcagtcaccc tgacgcaccc aatcaccaaa atcgataggg aggttctcta
     3601 ccaggagttc gatgagatgg aagagtgctc tcagcactat ccctacgatg tgcccgatta
     3661 cgctggcgcg ccttgattcc tgccctggct tggcctggac tcaccctgcc tccaccacct
     3721 gggcccttgg tctggactga atagcccaag cccttcgtac cctagccttt cttcccaccc
     3781 ttcttattta tttcctttct gactggatcg agcccattgg agggaggaca ctcctctctg
     3841 catgtatccc tgcaccccag atcagggccc ctagacccca ggaacctggg gtctgggggg
     3901 gagctgggct cctggttccg agcccttgct ccttaagact cctacccctg cccgccccca
     3961 tgcacacaca gacccactgg gggcctctgc cctccccatt ttctcccaca cacattccag
     4021 aagtcagggc ccctctcaag gagtacccgc tgtagggatg cagggccaca ggcttccgcg
     4081 ctctcccgag gcaggggctg gggtcacccc tgccccatca taactcccca cgccgtttga
     4141 gttctccttt attttctcca ttcttttctt ctcaaaggtg gtttttgggg ggagaagcag
     4201 gggggctctc ctgagggtcc ccccgtcttt cacacacctc ccaccttttt tctttgccgg
     4261 tttgcatgag tggaaggtct aactgtggct tttttttttt tctgggattt tatttcattt
     4321 tatttgattt tatttttttg ggaaaagggg agggatgggt ctggggagtg gggaatgtgg
     4381 gaggggggtg ggggggcaaa gggtcagggg ttgggtgtcc gggagccagg ggaggacagg
     4441 aaatgctgcc gccttctgca atttatttat ttttttcttt tgagagagtg aaaggaagag
     4501 acagacactt gaaaaaaaaa aaaaaagggc ggccgctcta gagtatccct cgaggggccc
     4561 aagcttacgc gtacccagct ttcttgtaca aagtggtccc tatagtgagt cgtattataa
     4621 gctaggcact ggccgtcgtt ttacaacgtc gtgactggga aaactgctag cttgggatct
     4681 ttgtgaagga accttacttc tgtggtgtga cataattgga caaactacct acagagattt
     4741 aaagctctaa ggtaaatata aaatttttaa gtgtataatg tgttaaacta gctgcatatg
     4801 cttgctgctt gagagttttg cttactgagt atgatttatg aaaatattat acacaggagc
     4861 tagtgattct aattgtttgt gtattttaga ttcacagtcc caaggctcat ttcaggcccc
     4921 tcagtcctca cagtctgttc atgatcataa tcagccatac cacatttgta gaggttttac
     4981 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     5041 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     5101 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     5161 atgtatctta tcatgtctgg atcgatcctg cattaatgaa tcggccaacg cgcggggaga
     5221 ggcggtttgc gtattggctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     5281 cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     5341 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     5401 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     5461 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     5521 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     5581 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     5641 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     5701 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     5761 atttcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     5821 atacattcaa atatgtatcc gctcatgcca ggtcttggac tggtgagaac ggcttgctcg
     5881 gcagcttcga tgtgtgctgg agggagaata aaggtctaag atgtgcgata gagggaagtc
     5941 gcattgaatt atgtgctgtg tagggatcgc tggtatcaaa tatgtgtgcc cacccctggc
     6001 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     6061 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     6121 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     6181 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     6241 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     6301 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     6361 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     6421 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     6481 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     6541 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     6601 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     6661 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     6721 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     6781 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     6841 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     6901 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     6961 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gccataactt
     7021 cgtataatgt atgctatacg aagttatggc atgaccaaaa tcccttaacg tgagttttcg
     7081 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     7141 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     7201 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     7261 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     7321 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     7381 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     7441 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     7501 tacctacagc gtgagcattg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     7561 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     7621 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     7681 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     7741 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     7801 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     7861 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc
     7921 cccgcgcgtt ggccgattca ttaatgcaga gcttgcaatt cgcgcgtttt tcaatattat
     7981 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     8041 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     8101 accattatta tcatgacatt aacctataaa aataggcgta gtacgaggcc ctttcactca
     8161 ttagatgcat gt
//
