LOCUS       sequence-368498-        5505 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 5505)
  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 5505)
  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-mCherry-6H
FEATURES             Location/Qualifiers
     source          1..5505
                     /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"
     protein_bind    19..43
                     /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             56..78
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     regulatory      488..497
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             494..1201
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEDDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGHHSTGGMDELYK"
     primer_bind     complement(639..657)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     907..926
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1101..1124
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             1208..1225
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(1255..1273)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1256..1273)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1331..1349)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1345..1392
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1628..1649)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1799..2881)
                     /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     2843..2862
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2882..2959)
                     /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     3102..3121
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3275..3294)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3394..3416
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3454..3472)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3540..3644
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3645..3713,3714..4505)
                     /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(3863..3882)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4676..5264
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5165..5184
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5418..5435
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgtc caacattata gttgagcatc agtattctca
      121 tagactgaaa ttgactgtgg tgagagctga aaatgtaact aaaggagcat ttggggattt
      181 attggacacc ccggaccctt atgttgagct gtcagtgcca accacaccag agtcaagaaa
      241 acgaacccgc cacattaaca atgacattaa ccccaagtgg aacgagacct ttgagttcat
      301 attggatccc aatcaatcca atgttttgga ggtgacactg atggatgcca attatgtcat
      361 ggatgaaact cttggcacag caaaatattc cctctcaaaa ctcaaagtgg cacaaatgga
      421 gcatgtgacc ctctctatcg gcaagacaac caaagtgttc ctcgacttgc tcttggaagg
      481 accggtcgcc accatggtga gcaagggcga ggatgacaac atggccatca tcaaggagtt
      541 catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg
      601 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
      661 cggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc
      721 ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt
      781 caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc
      841 ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc
      901 cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta
      961 ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg
     1021 ccactacgac gccgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg
     1081 cgcctacaac gtcaacatca agctggacat cacctcccac aacgaggact acaccatcgt
     1141 ggaacagtac gagcgcgccg agggccacca ctccaccggc ggcatggacg agctgtacaa
     1201 gtccggacat catcatcatc atcactgagc ggccgcatgc ataagcttga gtattctata
     1261 gtgtcaccta aatcccagct tgatccggct gctaacaaag cccgaaagga agctgagttg
     1321 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     1381 aggggttttt tgctgaaagg aggaactata tccggataac ctggcgtaat agcgaagagg
     1441 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg aaattgtaag
     1501 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     1561 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     1621 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     1681 gcgaaaaacc gtctatcagg gcgatggccc acagcttgca tgcctgcagg tcggaagcat
     1741 aaagtgtaaa gcctggggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     1801 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     1861 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     1921 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     1981 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     2041 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     2101 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     2161 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     2221 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     2281 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     2341 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     2401 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     2461 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     2521 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     2581 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     2641 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     2701 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     2761 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     2821 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     2881 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     2941 tgcaccattc gatggtgtcg ggtaccgagc tcgaattgtg aaccatcacc ctaatcaagt
     3001 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     3061 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     3121 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     3181 gcgcttaatg cgccgctaca gggcgcgtcc tgatgcggta ttttctcctt acgcatctgt
     3241 gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     3301 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     3361 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     3421 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     3481 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     3541 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     3601 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     3661 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     3721 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     3781 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     3841 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     3901 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     3961 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     4021 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     4081 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     4141 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     4201 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     4261 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     4321 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     4381 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     4441 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     4501 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     4561 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     4621 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4681 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4741 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4801 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     4861 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4921 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4981 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     5041 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     5101 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     5161 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5221 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     5281 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     5341 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     5401 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     5461 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctgat
//
