LOCUS       sequence-368500-        6035 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 6035)
  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 6035)
  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: pGEX-6P-1-hs-ALPS 1-ALPS2-mCherry
FEATURES             Location/Qualifiers
     source          1..6035
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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)."
     CDS             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..941
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     regulatory      1311..1320
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1317..2024
                     /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(1462..1480)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1730..1749
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1924..1947
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(2085..2107)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2252..2270)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2338..2442
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2443..2511,2512..3303)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2661..2680)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3474..4062
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3963..3982
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4216..4233
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4306..4383
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4384..5466
                     /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     complement(4403..4422)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5515..5520,5521..5538,5539..5545)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5553..5569
                     /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)."
     primer_bind     5577..5593
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5577..5593
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5589..6035,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5609..5626)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5609..5625)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5618..5640)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 agcttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gaagttctgt tccaggggcc cctgggatcc ccggaattca
      961 tgcctcagaa gaaagaagat gacttcctca acaacgccat gtcctccctg tactcgggct
     1021 ggagcagctt caccactgga gccagccggt ttgcctcggc agccaaggag ggcgctacaa
     1081 agtttggatc ccaagcgagt cagaaggcgt ccgagctggg ccacagcctg aacgagaacg
     1141 tcctcaagcc tgcgcaggag aaggtgaagg agggaaagat ttttgatgat gtctccagtg
     1201 gggtctctca gttggcgtcc aaggtccagg gagtcggtag taagggatgg cgggacgtca
     1261 ccaccttttt ttcggggaaa gcagagggcc ccttggacag cggaccggtc gccaccatgg
     1321 tgagcaaggg cgaggatgac aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc
     1381 acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
     1441 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggcggcccc ctgcccttcg
     1501 cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg
     1561 ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga
     1621 tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg
     1681 agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc
     1741 agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc
     1801 tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgccgagg
     1861 tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca
     1921 tcaagctgga catcacctcc cacaacgagg actacaccat cgtggaacag tacgagcgcg
     1981 ccgagggcca ccactccacc ggcggcatgg acgagctgta caaggcggcc gcatcgtgac
     2041 tgactgacga tctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     2101 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     2161 ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat
     2221 agcggagtgt ataattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt
     2281 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     2341 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     2401 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     2461 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     2521 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     2581 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     2641 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa
     2701 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     2761 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     2821 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     2881 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     2941 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca
     3001 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     3061 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     3121 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     3181 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     3241 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     3301 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     3361 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     3421 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     3481 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3541 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     3601 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     3661 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     3721 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     3781 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     3841 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3901 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3961 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4021 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4081 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4141 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     4201 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     4261 tttctcctta cgcatctgtg cggtatttca caccgcataa attccgacac catcgaatgg
     4321 cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg
     4381 aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc
     4441 gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa
     4501 gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa
     4561 cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt
     4621 gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta
     4681 gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc
     4741 agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc
     4801 tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt
     4861 attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac
     4921 cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct
     4981 ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac
     5041 tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc
     5101 actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag
     5161 tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc
     5221 tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc
     5281 agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg
     5341 cccgtctcac tggtgaaaag aaaaaccacg ctagcgccca atacgcaaac cgcctctccc
     5401 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     5461 cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcacccc aggctttaca
     5521 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
     5581 aaacagctat gaccatgatt acggattcac tggccgtcgt tttacaacgt cgtgactggg
     5641 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     5701 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     5761 aatggcgctt tgcctggttt ccggcaccag aagcggtgcc ggaaagctgg ctggagtgcg
     5821 atcttcctga ggccgatact gtcgtcgtcc cctcaaactg gcagatgcac ggttacgatg
     5881 cgcccatcta caccaacgtg acctatccca ttacggtcaa tccgccgttt gttcccacgg
     5941 agaatccgac gggttgttac tcgctcacat ttaatgttga tgaaagctgg ctacaggaag
     6001 gccagacgcg aattattttt gatggcgttg gaatt
//
