LOCUS       sequence-368501-        5535 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 5535)
  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 5535)
  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-cPla2-C2-EGFP-GH
FEATURES             Location/Qualifiers
     source          1..5535
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      77..665
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     566..585
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     819..836
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     907..926
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        907..925
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    925..949
                     /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             962..984
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     regulatory      1415..1424
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1421..1423,1424..1426,1427..2137)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1421..1423,1424..1426,1427..2137)
                     /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(1466..1487)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1727..1746)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2074..2095
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2144..2161
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(2191..2209)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2192..2209)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2267..2285)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2281..2328
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2564..2585)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2735..3817)
                     /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     3779..3798
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3818..3895)
                     /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     4038..4057
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4211..4230)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4330..4352
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4390..4408)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4476..4580
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4581..4649,4650..5441)
                     /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(4799..4818)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
       61 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
      121 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      181 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
      241 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      301 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      361 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      421 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      481 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      541 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      601 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      661 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      721 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
      781 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
      841 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
      901 gctgattaat acgactcact atagggaatt gtgagcggat aacaattcct ctagaaataa
      961 ttttgtttaa ctttaagaag gagatatatc catgggttca tttatagatc cttaccagca
     1021 cattatagtg gagcaccagt attcccacaa gtttacggta gtggtgttac gtgccaccaa
     1081 agtgacaaag ggggcctttg gtgacatgct tgatactcca gatccctatg tggaactttt
     1141 tatctctaca acccctgaca gcaggaagag aacaagacat ttcaataatg acataaaccc
     1201 tgtgtggaat gagacctttg aatttatttt ggatcctaat caggaaaatg ttttggagat
     1261 tacgttaatg gatgccaatt atgtcatgga tgaaactcta gggacagcaa catttactgt
     1321 atcttctatg aaggtgggag aaaagaaaga agttcctttt attttcaacc aagtcactga
     1381 aatggttcta gaaatgtctc ttgaaggacc ggtcgccacc atggtgagca agggcgagga
     1441 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     1501 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     1561 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     1621 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     1681 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     1741 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     1801 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     1861 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     1921 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     1981 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     2041 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     2101 cgccgggatc actctcggca tggacgagct gtacaagtcc ggacatcatc atcatcatca
     2161 ctgagcggcc gcatgcataa gcttgagtat tctatagtgt cacctaaatc ccagcttgat
     2221 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     2281 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     2341 actatatccg gataacctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     2401 agttgcgcag cctgaatggc gaatggaaat tgtaagcgtt aatattttgt taaaattcgc
     2461 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     2521 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     2581 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     2641 tggcccacag cttgcatgcc tgcaggtcgg aagcataaag tgtaaagcct ggggtgccta
     2701 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     2761 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     2821 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     2881 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     2941 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     3001 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     3061 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     3121 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     3181 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     3241 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     3301 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     3361 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     3421 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     3481 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     3541 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     3601 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     3661 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     3721 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     3781 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     3841 cttccgggcg ctatcatgcc ataccgcgaa aggttttgca ccattcgatg gtgtcgggta
     3901 ccgagctcga attgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     3961 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     4021 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     4081 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     4141 gcgtcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt
     4201 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     4261 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     4321 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     4381 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     4441 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     4501 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     4561 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     4621 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     4681 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     4741 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     4801 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     4861 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     4921 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     4981 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     5041 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     5101 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     5161 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     5221 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     5281 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     5341 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     5401 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     5461 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     5521 tcctttttga taatc
//
