LOCUS       sequence-368507-        5565 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 5565)
  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 5565)
  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-AKT1-PH-EGFP-6H
FEATURES             Location/Qualifiers
     source          1..5565
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(32..100,101..892)
                     /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(250..269)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1063..1651
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1552..1571
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1805..1822
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1893..1912
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1893..1911
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1911..1935
                     /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             1948..1970
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     regulatory      2431..2440
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2437..2439,2440..2442,2443..3153)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2437..2439,2440..2442,2443..3153)
                     /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(2482..2503)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2743..2762)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3090..3111
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3160..3177
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(3207..3225)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3208..3225)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3283..3301)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3297..3344
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3580..3601)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3751..4833)
                     /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     4795..4814
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4834..4911)
                     /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     5054..5073
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(5227..5246)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5346..5368
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5406..5424)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(5492..5565,1..31)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
       61 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
      121 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
      181 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
      241 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
      301 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
      361 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
      421 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
      481 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
      541 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
      601 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
      661 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
      721 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
      781 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
      841 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
      901 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
      961 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     1021 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     1081 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     1141 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     1201 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     1261 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     1321 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     1381 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     1441 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     1501 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     1561 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     1621 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     1681 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     1741 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     1801 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     1861 cgcgcgttgg ccgattcatt aatgcagctg attaatacga ctcactatag ggaattgtga
     1921 gcggataaca attcctctag aaataatttt gtttaacttt aagaaggaga tatatccatg
     1981 agcgacgtgg ctattgtgaa ggagggttgg ctgcacaaac gaggggagta catcaagacc
     2041 tggcggccac gctacttcct cctcaagaat gatggcacct tcattggcta caaggagcgg
     2101 ccgcaggatg tggaccaacg tgaggctccc ctcaacaact tctctgtggc gcagtgccag
     2161 ctgatgaaga cggagcggcc ccggcccaac accttcatca tccgctgcct gcagtggacc
     2221 actgtcatcg aacgcacctt ccatgtggag actcctgagg agcgggagga gtggacaacc
     2281 gccatccaga ctgtggctga cggcctcaag aagcaggagg aggaggagat ggacttccgg
     2341 tcgggctcac ccagtgacaa ctcaggggct gaagagatgg aggtgtccct ggccaagccc
     2401 aagcaccgcg tgaccatgaa cggaccggtc gccaccatgg tgagcaaggg cgaggagctg
     2461 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     2521 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     2581 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     2641 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     2701 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     2761 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     2821 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     2881 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     2941 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     3001 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     3061 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     3121 gggatcactc tcggcatgga cgagctgtac aagtccggac atcatcatca tcatcactga
     3181 gcggccgcat gcataagctt gagtattcta tagtgtcacc taaatcccag cttgatccgg
     3241 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
     3301 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
     3361 tatccggata acctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     3421 gcgcagcctg aatggcgaat ggaaattgta agcgttaata ttttgttaaa attcgcgtta
     3481 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     3541 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     3601 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     3661 ccacagcttg catgcctgca ggtcggaagc ataaagtgta aagcctgggg tgcctaatga
     3721 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3781 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3841 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     3901 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     3961 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     4021 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     4081 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     4141 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     4201 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     4261 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     4321 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     4381 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     4441 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     4501 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     4561 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     4621 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     4681 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     4741 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     4801 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     4861 cgggcgctat catgccatac cgcgaaaggt tttgcaccat tcgatggtgt cgggtaccga
     4921 gctcgaattg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     4981 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     5041 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     5101 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     5161 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac
     5221 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     5281 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     5341 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     5401 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     5461 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     5521 aatacattca aatatgtatc cgctcatgag acaataaccc tgata
//
