LOCUS       sequence-343238-        4887 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4887)
  AUTHORS   Violin JD, Zhang J, Tsien RY, Newton AC
  TITLE     A genetically encoded fluorescent reporter reveals oscillatory 
            phosphorylation by protein kinase C.
  JOURNAL   J Cell Biol. 2003 Jun 9. 161(5):899-909.
  PUBMED    12782683
REFERENCE   2  (bases 1 to 4887)
  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: "J Cell 
            Biol. 2003 Jun 9. 161(5):899-909."
COMMENT     Sequence Label: pRSET B-CKAR
FEATURES             Location/Qualifiers
     source          1..4887
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             51..73
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             93..110
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             114..146
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(150..158,159..173)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(183..185,186..188,189..899)
                     /codon_start=1
                     /product="cyan variant of GFP"
                     /label=CFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     CDS             join(1458..1460,1461..2174)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2269..2287)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2283..2330
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2427..2882
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2514..2533)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2724..2745
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2908..3012
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3013..3081,3082..3873)
                     /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(3231..3250)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4044..4632
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4533..4552
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4786..4803
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg agaccacaac ggtttccctc tagaaataat tttgtttaac
       61 tttaagaagg agatatacat atgcggggtt ctcatcatca tcatcatcat ggtatggcta
      121 gcatgactgg tggacagcaa atgggtcggg atctgtacga cgatgacgat aaggatctca
      181 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
      241 acggcgacgt aaacggccac aggttcagcg tgtccggcga gggcgagggc gatgccacct
      301 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
      361 ccctcgtgac caccctgacc tggggcgtgc agtgcttcag ccgctacccc gaccacatga
      421 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgtaccatct
      481 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
      541 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
      601 acaagctgga gtacaactac atcagccaca acgtctatat caccgccgac aagcagaaga
      661 acggcatcaa ggcccacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
      721 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
      781 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
      841 tcctgctgga gttcgtgacc gccgccggga tcactcacgg catggacgag ctgtacaagg
      901 ctggtaccaa gggtaatggt aggtttttaa ctttaaaacc attgcctgac agcattattc
      961 aagaaagcct ggagattcag caaggtgtga atccattttt cattggtaga tccgaggatt
     1021 gcaattgtaa aattgaagac agtaggttgt ctcgagttca ttgcttcatt ttcaaaaaga
     1081 ggcatgctgt aggcaaaagc atgtatgaat ctccggcaca aggtttagat gatatttggt
     1141 attgccacac cggaactaac gtgagctatt taaataataa ccgcatgata cagggtacga
     1201 aattcctttt acaagacgga gatgaaatca agatcatttg ggataaaaac aataaatttg
     1261 tcattggctt taaagtggaa attaacgata ctacaggtct gtttaacgag ggattaggta
     1321 tgttacaaga acaaagagta gtacttaagc aaacagccga agaaaaagat ttggtgaaaa
     1381 agttaggagg atccggaggt cgattcagaa gattccagac gttgaagatc aaggctaagg
     1441 caggtggatc tggtggagtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     1501 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     1561 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     1621 tgccctggcc caccctcgtg accaccttcg gctacggcct gaagtgcttc gcccgctacc
     1681 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     1741 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     1801 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     1861 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     1921 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     1981 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     2041 tgcccgacaa ccactacctg agctaccagt ccgccctgag caaagacccc aacgagaagc
     2101 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactcac ggcatggacg
     2161 agctgtacaa gtaatctaga ggtcgagatc tgcagctggt accatggaat tcgaagcttg
     2221 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     2281 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     2341 gaactatatc cggatctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     2401 gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     2461 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2521 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2581 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2641 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2701 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2761 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     2821 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     2881 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     2941 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     3001 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     3061 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     3121 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     3181 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     3241 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     3301 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     3361 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     3421 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     3481 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     3541 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     3601 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     3661 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     3721 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     3781 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     3841 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     3901 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     3961 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     4021 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     4081 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     4141 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     4201 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     4261 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4321 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     4381 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     4441 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     4501 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     4561 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     4621 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     4681 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     4741 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     4801 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     4861 taatgcagga tctcgatccc gcgaaat
//
