LOCUS       sequence-368504-        5520 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein Expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5520)
  AUTHORS   Shen Z, Belcheva KT, Jelcic M, Hui KL, Katikaneni A, Niethammer P
  TITLE     A synergy between mechanosensitive calcium- and membrane-binding 
            mediates tension-sensing by C2-like domains.
  JOURNAL   Proc Natl Acad Sci U S A. 2022 Jan 4;119(1). pii: 2112390119. doi: 
            10.1073/pnas.2112390119.
  PUBMED    34969839
REFERENCE   2  (bases 1 to 5520)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2022-01-4"; volume: "119(1). pii: 
            2112390119. doi"; pages: " 10.1073/pnas.2112390119"
COMMENT     Sequence Label: Phat2-hs-PKCgamma-C2-EGFP-6H
FEATURES             Location/Qualifiers
     source          1..5520
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(32..100,101..892)
                     /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(250..269)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1063..1651
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1552..1571
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1805..1822
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1893..1912
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1893..1911
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1911..1935
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             1948..1970
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     regulatory      2386..2395
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2392..2394,2395..2397,2398..3108)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2392..2394,2395..2397,2398..3108)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2437..2458)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2698..2717)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3045..3066
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3115..3132
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(3162..3180)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3163..3180)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3238..3256)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3252..3299
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3535..3556)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3706..4788)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4750..4769
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4789..4866)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     5009..5028
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(5182..5201)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5301..5323
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5361..5379)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(5447..5520,1..31)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
       61 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
      121 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
      181 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
      241 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
      301 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
      361 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
      421 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
      481 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
      541 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
      601 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
      661 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
      721 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
      781 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
      841 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
      901 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
      961 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     1021 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     1081 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     1141 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     1201 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     1261 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     1321 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     1381 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     1441 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     1501 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     1561 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     1621 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     1681 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     1741 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     1801 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     1861 cgcgcgttgg ccgattcatt aatgcagctg attaatacga ctcactatag ggaattgtga
     1921 gcggataaca attcctctag aaataatttt gtttaacttt aagaaggaga tatatccatg
     1981 cgcgggcgcc tgcagctgga gatccgggct cccacagcag atgagatcca cgtaactgtt
     2041 ggcgaggccc gtaacctaat tcctatggac cccaatggtc tctctgatcc ctatgtgaaa
     2101 ctgaagctca tcccagaccc tcggaacctg acgaaacaga agacccgaac ggtgaaagcc
     2161 acgctaaacc ctgtgtggaa tgagaccttt gtgttcaacc tgaagccagg ggatgtggag
     2221 cgccggctca gcgtggaggt gtgggactgg gaccggacct cccgcaacga cttcatgggg
     2281 gccatgtcct ttggcgtctc ggagctgctc aaggcgcccg tggatggctg gtacaagtta
     2341 ctgaaccagg aggagggcga gtattacaat gtgccgggac cggtcgccac catggtgagc
     2401 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     2461 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     2521 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     2581 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     2641 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     2701 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     2761 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     2821 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     2881 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     2941 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     3001 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     3061 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagtc cggacatcat
     3121 catcatcatc actgagcggc cgcatgcata agcttgagta ttctatagtg tcacctaaat
     3181 cccagcttga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
     3241 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     3301 tgaaaggagg aactatatcc ggataacctg gcgtaatagc gaagaggccc gcaccgatcg
     3361 cccttcccaa cagttgcgca gcctgaatgg cgaatggaaa ttgtaagcgt taatattttg
     3421 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     3481 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     3541 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     3601 tatcagggcg atggcccaca gcttgcatgc ctgcaggtcg gaagcataaa gtgtaaagcc
     3661 tggggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     3721 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     3781 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     3841 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     3901 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     3961 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     4021 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     4081 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     4141 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     4201 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     4261 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     4321 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     4381 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     4441 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     4501 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     4561 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     4621 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     4681 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     4741 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     4801 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc accattcgat
     4861 ggtgtcgggt accgagctcg aattgtgaac catcacccta atcaagtttt ttggggtcga
     4921 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     4981 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     5041 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     5101 cgctacaggg cgcgtcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     5161 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     5221 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     5281 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     5341 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     5401 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     5461 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
//
