LOCUS       sequence-368505-        5472 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 5472)
  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 5472)
  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-Alox12-PLAT-EGFP-6H
FEATURES             Location/Qualifiers
     source          1..5472
                     /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      2211..2220
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2217..2219,2220..2222,2223..2933)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2217..2219,2220..2222,2223..2933)
                     /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(2262..2283)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2523..2542)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2870..2891
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2940..2957
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(2987..3005)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2988..3005)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3063..3081)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3077..3124
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3360..3381)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3531..4613)
                     /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     4575..4594
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4614..4691)
                     /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     4834..4853
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(5007..5026)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5126..5148
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5186..5204)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5272..5376
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5377..5445,5446..5472,1..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 atggagtaca
     1861 aagtgacagt ggccaccggg acttctgaat attcagggac caataattat gtgtacgtga
     1921 ctctgattgg agagaaagga aagagcgaga ggacgctgct ggacaatcct ggcctggact
     1981 tctgcagagg agctgtggat gattacatag ttaaaagcga tactgatttg ggccccctga
     2041 ttctggtgcg actggagaag cacaagtact tcatagaaga caactggttc tgccgctacg
     2101 tgaaagtcag tgtacctgga gaaagatgtt gttacacatt tccttgttac cgctggcttg
     2161 tgggagatgg agtgtttgta gagctcagag aaggcacagc cggaccggtc gccaccatgg
     2221 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     2281 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     2341 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     2401 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     2461 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     2521 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     2581 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     2641 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     2701 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     2761 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     2821 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     2881 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtccggac
     2941 atcatcatca tcatcactga gcggccgcat gcataagctt gagtattcta tagtgtcacc
     3001 taaatcccag cttgatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     3061 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     3121 tttgctgaaa ggaggaacta tatccggata acctggcgta atagcgaaga ggcccgcacc
     3181 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggaaattgta agcgttaata
     3241 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     3301 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     3361 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     3421 ccgtctatca gggcgatggc ccacagcttg catgcctgca ggtcggaagc ataaagtgta
     3481 aagcctgggg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     3541 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     3601 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     3661 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     3721 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     3781 tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg
     3841 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     3901 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     3961 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     4021 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     4081 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     4141 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     4201 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     4261 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     4321 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     4381 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     4441 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     4501 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     4561 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     4621 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcaccat
     4681 tcgatggtgt cgggtaccga gctcgaattg tgaaccatca ccctaatcaa gttttttggg
     4741 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     4801 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     4861 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     4921 tgcgccgcta cagggcgcgt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4981 tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     5041 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     5101 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     5161 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     5221 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     5281 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     5341 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     5401 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     5461 gtgaaagtaa aa
//
