LOCUS       sequence-368508-        5514 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 5514)
  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 5514)
  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-dr-cPla2-C2-L32A-L33A-EGFP-6H
FEATURES             Location/Qualifiers
     source          1..5514
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(123..142)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      936..1524
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1425..1444
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1678..1695
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1766..1785
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1766..1784
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1784..1808
                     /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             1821..1843
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     regulatory      2253..2262
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2259..2261,2262..2264,2265..2975)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2259..2261,2262..2264,2265..2975)
                     /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(2304..2325)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2565..2584)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2912..2933
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2982..2999
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(3029..3047)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3030..3047)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3105..3123)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3119..3166
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3402..3423)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3573..4655)
                     /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     4617..4636
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4656..4733)
                     /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     4876..4895
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(5049..5068)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5168..5190
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5228..5246)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5314..5418
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5419..5487,5488..5514,1..765)
                     /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"
ORIGIN
        1 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
       61 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
      121 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
      181 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
      241 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
      301 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
      361 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
      421 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
      481 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
      541 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
      601 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
      661 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
      721 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
      781 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
      841 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
      901 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
      961 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     1021 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     1081 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     1141 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     1201 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     1261 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     1321 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     1381 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     1441 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     1501 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     1561 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     1621 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     1681 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     1741 tggccgattc attaatgcag ctgattaata cgactcacta tagggaattg tgagcggata
     1801 acaattcctc tagaaataat tttgtttaac tttaagaagg agatatatcc atgtccaaca
     1861 ttatagttga gcatcagtat tctcatagac tgaaattgac tgtggtgaga gctgaaaatg
     1921 taactaaagg agcatttggg gatgcagcgg acaccccgga cccttatgtt gagctgtcag
     1981 tgccaaccac accagagtca agaaaacgaa cccgccacat taacaatgac attaacccca
     2041 agtggaacga gacctttgag ttcatattgg atcccaatca atccaatgtt ttggaggtga
     2101 cactgatgga tgccaattat gtcatggatg aaactcttgg cacagcaaaa tattccctct
     2161 caaaactcaa agtggcacaa atggagcatg tgaccctctc tatcggcaag acaaccaaag
     2221 tgttcctcga cttgctcttg gaaggaccgg tcgccaccat ggtgagcaag ggcgaggagc
     2281 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     2341 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     2401 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     2461 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     2521 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     2581 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     2641 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     2701 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     2761 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     2821 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
     2881 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     2941 ccgggatcac tctcggcatg gacgagctgt acaagtccgg acatcatcat catcatcact
     3001 gagcggccgc atgcataagc ttgagtattc tatagtgtca cctaaatccc agcttgatcc
     3061 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     3121 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     3181 tatatccgga taacctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     3241 ttgcgcagcc tgaatggcga atggaaattg taagcgttaa tattttgtta aaattcgcgt
     3301 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     3361 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     3421 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     3481 gcccacagct tgcatgcctg caggtcggaa gcataaagtg taaagcctgg ggtgcctaat
     3541 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3601 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3661 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     3721 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     3781 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     3841 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     3901 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     3961 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     4021 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     4081 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     4141 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     4201 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     4261 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     4321 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     4381 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     4441 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     4501 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     4561 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     4621 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     4681 tccgggcgct atcatgccat accgcgaaag gttttgcacc attcgatggt gtcgggtacc
     4741 gagctcgaat tgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     4801 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     4861 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     4921 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     4981 gtcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc
     5041 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     5101 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     5161 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     5221 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     5281 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     5341 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     5401 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     5461 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaa
//
