LOCUS       pEMJ_042                4212 bp ds-DNA     circular     15-OCT-2023
DEFINITION  .
FEATURES             Location/Qualifiers
     primer_bind     154..171
                     /label="L4440"
                     /note="note: L4440 vector, forward primer"
     promoter        256..274
                     /label="T7 promoter"
                     /note="note: promoter for bacteriophage T7 RNA polymerase"
     primer_bind     256..275
                     /label="T7"
                     /note="note: T7 promoter, forward primer"
     misc_feature    256..280
                     /label="EMJ_P305"
     RBS             306..328
                     /label="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(336..1682,1683..1685)
                     /translation="MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNKTGHAVRAIGRLSSLENVYITADKQKNGIKANFHVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTKLSKDPNEKRDHMVLLEFVTAAGITHGMDELYGGTGGSASQGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFISTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDPDGDGTIDTKELGTVMRSLGQNPTEAELQDMINEVDPDGDGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDPDGDGYIDAAELRHVMTNLGEKLTDEEVDEMIREADPDGDGQVDYEEFVQMMTAK*"
     primer_bind     complement(341..365)
                     /label="EMJ_P340"
     CDS             348..365
                     /label="6xHis"
                     /note="product: 6xHis affinity tag translation: HHHHHH"
     CDS             372..401
                     /label="T7 tag (gene 10 leader)"
                     /note="product: leader peptide from bacteriophage T7 gene 10 note: promotes efficient translation in E. coli translation: MASMTGGQQMG"
     misc_feature    404..428
                     /label="EMJ_P330"
     CDS             405..428
                     /label="Xpress(TM) tag"
                     /note="product: Xpress(TM) epitope tag, including an enterokinaserecognition and cleavage site translation: DLYDDDDK"
     misc_feature    405..428
                     /label="EMJ_P327"
     misc_feature    complement(405..428)
                     /label="EMJ_P099"
     misc_feature    complement(405..428)
                     /label="EMJ_P325"
     misc_feature    435..444
                     /label="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)"
                     /note="regulatory_class: other"
     CDS             456..512
                     /label="calmodulin-binding peptide"
                     /note="product: calmodulin-binding peptide from avian smooth muscle myosin light chain kinase translation: RRKWNKTGHAVRAIGRLSS"
     misc_feature    513..518
                     /label="L1"
     misc_feature    519..527
                     /label="Mutational region?"
     misc_feature    519..785
                     /label="sfGFP C-terminus"
     misc_feature    582..584
                     /label="Mutation region?"
     misc_feature    651..653
                     /label="Mutation region?"
     misc_feature    672..674
                     /label="Mutation region?"
     misc_feature    765..767
                     /label="Mutation region?"
     misc_feature    804..1235
                     /label="sfGFP N-terminus"
     CDS             804..1235
                     /label="sfGFP N-terminus"
                     /translation="ASQGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFISTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYN"
     misc_feature    808..828
                     /label="EMJ_P342"
     misc_feature    complement(808..828)
                     /label="EMJ_P341"
     misc_feature    834..836
                     /label="Mutation region?"
     misc_feature    1032..1034
                     /label="T78H"
     misc_feature    1214..1235
                     /label="End sfGFP N terminus"
     misc_feature    complement(1214..1241)
                     /label="EMJ_P095"
     CDS             1236..1241
                     /label="GCaMP6s"
                     /note="transl_table: 1 codon_start: 1 translation: MGS,HHHHHH,G,MASMTGGQQMG,R,DLYDDDDK,DLATMVDSS,RRKWNKTGHAVRVIGRLSS,LE,NVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK,GGTGGS,MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN,LP,DQLTEEQIAEFKELFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGNGYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK product: improved fluorescent protein-based calcium sensor (Chen et al., 2013)"
     misc_feature    1236..1241
                     /label="L2"
     misc_feature    1242..1260
                     /label="EMJ_P363"
     misc_feature    complement(1242..1262)
                     /label="EMJ_P365"
     misc_feature    1242..1685
                     /label="Calmodulin (EF hand motif modifications)"
     misc_feature    1275..1283
                     /label="Mutation region"
     misc_feature    1281..1283
                     /label="Potential Mutation site (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3777791/)"
     misc_feature    1296..1331
                     /label="EF Hand -1"
     misc_feature    1299..1301
                     /label="K -> P mutation"
     misc_feature    1320..1322
                     /label="T to D mutation"
     misc_feature    complement(1396..1415)
                     /label="P090"
     misc_feature    1404..1439
                     /label="EF Hand 2"
     misc_feature    1407..1409
                     /label="A to P mutation"
     misc_feature    1416..1418
                     /label="N to D mutation"
     misc_feature    complement(1445..1469)
                     /label="EMJ_P328"
     misc_feature    1458..1493
                     /label="Mutation region?"
     misc_feature    1458..1493
                     /label="CBP interface site?"
     misc_feature    1470..1472
                     /label="possible location for alteration to Y"
     misc_feature    1473..1495
                     /label="EMJ_P329"
     misc_feature    1506..1508
                     /label="possible location for alteration to G"
     misc_feature    1509..1531
                     /label="P091"
     misc_feature    1515..1550
                     /label="EF Hand 3"
     misc_feature    1518..1520
                     /label="A to P mutation"
     misc_feature    1518..1520
                     /label="K to P mutation"
     misc_feature    1527..1529
                     /label="N to D mutation"
     misc_feature    1539..1541
                     /label="S to D mutation"
     misc_feature    1623..1658
                     /label="EF Hand 4"
     misc_feature    1626..1628
                     /label="I to P mutation"
     misc_feature    1647..1649
                     /label="N to D mutation"
     primer          1686..1708
                     /label="EMJ_P089"
                     /note="sequence: gcgactctagatcataatcagcc"
     misc_feature    complement(1686..1708)
                     /label="EMJ_P316"
     primer_bind     complement(1849..1867)
                     /label="T7 Term"
                     /note="note: T7 terminator, reverse primer"
     terminator      1863..1910
                     /label="T7 terminator"
                     /note="note: transcription terminator for bacteriophage T7 RNA polymerase"
     rep_origin      2007..2462
                     /label="f1 ori"
                     /note="direction: RIGHT note: f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis"
     primer_bind     complement(2094..2113)
                     /label="F1ori-R"
                     /note="note: F1 origin, reverse primer"
     primer_bind     2304..2325
                     /label="F1ori-F"
                     /note="note: F1 origin, forward primer"
     misc_feature    complement(2468..2487)
                     /label="EMJ_P335"
     promoter        2488..2592
                     /label="AmpR promoter"
                     /note="gene: bla"
     CDS             2593..3453
                     /label="AmpR"
                     /note="gene: bla product: beta-lactamase note: confers resistance to ampicillin, carbenicillin, andrelated antibiotics translation: MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW"
     CDS             2593..3453
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW*"
     primer_bind     complement(2811..2830)
                     /label="Amp-R"
                     /note="note: Ampicillin resistance gene, reverse primer"
     rep_origin      3624..4212
                     /label="ori"
                     /note="direction: RIGHT note: high-copy-number ColE1/pMB1/pBR322/pUC origin of replication"
     primer_bind     4113..4132
                     /label="pBR322ori-F"
                     /note="note: pBR322 origin, forward primer"
ORIGIN
        1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
       61 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
      121 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
      181 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcaggatc
      241 tcgatcccgc gaaattaata cgactcacta tagggagacc acaacggttt ccctctagaa
      301 ataattttgt ttaactttaa gaaggagata tacatatgcg gggttctcat catcatcatc
      361 atcatggtat ggctagcatg actggtggac agcaaatggg tcgggatctg tacgacgatg
      421 acgataagga tctcgccacc atggtcgact catcacgtcg taagtggaat aagacaggtc
      481 acgcagtcag agctataggt cggctgagct cactcgagaa cgtgtatatt accgcggata
      541 aacagaaaaa cggcattaaa gcgaactttc acgtgcgcca taacgtggaa gatggcagcg
      601 tgcagctggc ggatcattat cagcagaaca ccccgattgg cgatggcccg gtgctgctgc
      661 cggataacca ttatctgagc acccagacca agctgagcaa agatccgaac gaaaaacgcg
      721 atcacatggt gctgctggaa tttgtgaccg cagcgggcat tacacacggc atggatgaac
      781 tgtatggcgg caccggcggc agcgcgagcc agggcgaaga actgtttacc ggcgtggtgc
      841 cgattctggt ggaactggat ggcgatgtga acggccataa atttagcgtg cgcggcgaag
      901 gcgaaggcga tgcgaccatt ggcaaactga ccctgaaatt tatttccacc accggcaaac
      961 taccggtgcc gtggccgacc ctggtgacca ccttaaccta tggcgtgcag tgctttagcc
     1021 gctatccgga tcatatgaaa cgccatgatt tttttaaaag cgcgatgccg gaaggctatg
     1081 tgcaggaacg caccattagc tttaaagatg atggcaaata taaaacccgc gcggtggtga
     1141 aatttgaagg cgataccctg gtgaaccgca ttgaactgaa aggcaccgat tttaaagaag
     1201 atggcaacat tctggggcat aaactggaat ataaccttcc tgaccaactg actgaagagc
     1261 agatcgcaga atttaaagag gctttctccc tatttgaccc ggacggggat gggacaataG
     1321 ATaccaagga gctggggacg gtgatgcggt ctctggggca gaaccccaca gaagcagagc
     1381 tgcaggacat gatcaatgaa gtagatCCGg acggtGATgg cacaatcgac ttccctgagt
     1441 tcctgacaat gatggcaaga aaaatgaaag acacggactc ggaagaagaa attagagaag
     1501 cgttcagggt gtttgatCCG gatggcGATg gctacatcGA Cgcagcagag cttcgccacg
     1561 tgatgacaaa ccttggagag aagttaacag atgaagaggt tgatgaaatg atcagggaag
     1621 cagacCCGga tggggatggt caggtaGATt acgaagagtt tgtacaaatg atgacagcga
     1681 agTAAgcgac tctagatcat aatcagccat accacatttg tagaggtttt acttgcttta
     1741 aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tcgaagcttg
     1801 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     1861 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     1921 gaactatatc cggatctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     1981 gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     2041 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2101 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2161 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2221 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2281 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2341 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     2401 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     2461 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     2521 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     2581 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     2641 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     2701 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     2761 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     2821 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     2881 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     2941 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     3001 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     3061 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     3121 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     3181 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     3241 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     3301 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     3361 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     3421 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     3481 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     3541 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     3601 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     3661 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     3721 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     3781 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     3841 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     3901 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     3961 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     4021 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     4081 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     4141 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     4201 agcctatgga aa
//