LOCUS       sequence-368533-        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-Y90A-V91A-EGFP-6H
FEATURES             Location/Qualifiers
     source          1..5514
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(66..134,135..926)
                     /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(284..303)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1097..1685
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1586..1605
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1839..1856
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1927..1946
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1927..1945
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1945..1969
                     /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             1982..2004
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     regulatory      2414..2423
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2420..2422,2423..2425,2426..3136)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2420..2422,2423..2425,2426..3136)
                     /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(2465..2486)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2726..2745)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3073..3094
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3143..3160
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(3190..3208)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3191..3208)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3266..3284)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3280..3327
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3563..3584)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3734..4816)
                     /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     4778..4797
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4817..4894)
                     /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     5037..5056
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(5210..5229)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5329..5351
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5389..5407)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(5475..5514,1..65)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
       61 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
      121 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
      181 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
      241 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
      301 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
      361 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
      421 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
      481 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
      541 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
      601 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
      661 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
      721 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
      781 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
      841 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
      901 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
      961 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     1021 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     1081 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     1141 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     1201 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     1261 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     1321 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     1381 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     1441 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     1501 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     1561 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     1621 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     1681 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     1741 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     1801 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     1861 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     1921 gctgattaat acgactcact atagggaatt gtgagcggat aacaattcct ctagaaataa
     1981 ttttgtttaa ctttaagaag gagatatatc catgtccaac attatagttg agcatcagta
     2041 ttctcataga ctgaaattga ctgtggtgag agctgaaaat gtaactaaag gagcatttgg
     2101 ggatttattg gacaccccgg acccttatgt tgagctgtca gtgccaacca caccagagtc
     2161 aagaaaacga acccgccaca ttaacaatga cattaacccc aagtggaacg agacctttga
     2221 gttcatattg gatcccaatc aatccaatgt tttggaggtg acactgatgg atgccaatgc
     2281 tgccatggat gaaactcttg gcacagcaaa atattccctc tcaaaactca aagtggcaca
     2341 aatggagcat gtgaccctct ctatcggcaa gacaaccaaa gtgttcctcg acttgctctt
     2401 ggaaggaccg gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     2461 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     2521 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     2581 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     2641 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     2701 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     2761 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     2821 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     2881 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     2941 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     3001 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     3061 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     3121 ggacgagctg tacaagtccg gacatcatca tcatcatcac tgagcggccg catgcataag
     3181 cttgagtatt ctatagtgtc acctaaatcc cagcttgatc cggctgctaa caaagcccga
     3241 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     3301 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg ataacctggc
     3361 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     3421 aatggaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     3481 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     3541 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     3601 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccacagc ttgcatgcct
     3661 gcaggtcgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aacttacatt
     3721 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     3781 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     3841 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     3901 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     3961 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg
     4021 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     4081 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     4141 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     4201 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     4261 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     4321 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     4381 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     4441 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     4501 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     4561 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     4621 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     4681 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     4741 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     4801 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     4861 taccgcgaaa ggttttgcac cattcgatgg tgtcgggtac cgagctcgaa ttgtgaacca
     4921 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     4981 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     5041 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     5101 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcctgatg cggtattttc
     5161 tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct
     5221 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     5281 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     5341 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     5401 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     5461 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaa
//
