LOCUS       sequence-456036-        3894 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Plasmid that encodes a B. Subtilis lipase (CLIP-BsLipA-eGFP) gene 
            fusion with the S77A catalytic mutant. Compatible with PURExpress 
            for in vitro transcription/translation. Chloramp.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3894)
  AUTHORS   Thompson, S, Zhang, Y, Yang, Z, Nichols,  LA, Fordyce, PM.
  TITLE     FACS-Sortable Microreactors for Ultra High-throughput Screening of 
            Catalysts in Biphasic Environments
  JOURNAL   bioRxiv.
REFERENCE   2  (bases 1 to 3894)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv."
COMMENT     Sequence Label: SMT503 - S77A
FEATURES             Location/Qualifiers
     source          1..3894
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(183..277)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(298..957)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGIMLNELQQYCDEWQGGA"
     primer_bind     872..891
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     1095..1114
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1095..1113
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1138..1683
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="optimized for bacterial expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     CDS             2287..3000
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             2287..2997
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     primer_bind     complement(2326..2347)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2934..2955
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3033..3053
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      3054..3111
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     primer_bind     complement(3135..3152)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3306..3894)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3386..3405)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ctcgaggctt
      181 ggattctcac caataaaaaa cgcccggcgg caaccgagcg ttctgaacaa atccagatgg
      241 agttatgagg tcattactgg atctatcaac aggagtccaa gcgagctcga tatcaaatta
      301 cgccccgccc tgccactcat cgcagtactg ttgtaattca ttaagcatta tgccgacatg
      361 gaagccatca caaacggcat gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc
      421 ttgcgtataa tatttgccca tggtgaaaac gggggcgaag aagttgtcca tattggccac
      481 gtttaaatca aaactggtga aactcaccca gggattggct gagacgaaaa acatattctc
      541 aataaaccct ttagggaaat aggccaggtt ttcaccgtaa cacgccacat cttgcgaata
      601 tatgtgtaga aactgccgga aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc
      661 agtttgctca tggaaaacgg tgtaacaagg gtgaacacta tcccatatca ccagctcacc
      721 gtctttcatt gccatacgaa attccggatg agcattcatc aggcgggcaa gaatgtgaat
      781 aaaggccgga taaaacttgt gcttattttt ctttacggtc tttaaaaagg ccgtaatatc
      841 cagctgaacg gtctggttat aggtacattg agcaactgac tgaaatgcct caaaatgttc
      901 tttacgatgc cattgggata tatcaacggt ggtatatcca gtgatttttt tctccatttt
      961 agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta gtgatcttat
     1021 ttcattatgg tgaaagttgg aacctcttac gtgcccgatc aactcgagtg ccacctgacg
     1081 tctaagaaac cgaataatac gactcactat agggcttaag tataaggaga tatacatatg
     1141 gacaaagatt gcgaaatgaa acgtaccacc ctggatagcc cgctgggcaa actggaactg
     1201 agcggctgcg aacagggcct gcatgaaatt aaactgctgg gtaaaggcac cagcgcggcc
     1261 gatgcggttg aagttccggc cccggccgcc gtgctgggtg gtccggaacc gctgatgcag
     1321 gcgaccgcgt ggctgaacgc gtattttcat cagccggaag cgattgaaga atttccggtt
     1381 ccggcgctgc atcatccggt gtttcagcag gagagcttta cccgtcaggt gctgtggaaa
     1441 ctgctgaaag tggttaaatt tggcgaagtg attagctatc agcagctggc ggccctggcg
     1501 ggtaatccgg ctgccaccgc cgccgttaaa accgcgctga gcggtaaccc ggtgccgatt
     1561 ctgattccgt gccatcgtgt ggttagctct agcggtgcgg ttggcggtta tgaaggtggt
     1621 ctggcggtga aagagtggct gctggcccat gaaggtcatc gtctgggtaa accgggtctg
     1681 ggaggtggag ggtctggggg aggaggctca ggcgcggaac ataacccggt ggtgatggtg
     1741 catggcattg gcggtgcgag ctttaacttt gcgggcatta aatcttatct ggtgagccag
     1801 ggctggagcc agaacgatct gtatgcggtg gacttttggg ataaaaccgg taccaactat
     1861 aacaacggcc cggtgctgag ccgttttgtg cagaaagtgc tggacgaaac cggcgcgaag
     1921 aaagtggata tcgtggcgca tgcgatgggt ggcgcgaaca ccctgtatta tattaagaac
     1981 ctggacggcg gcaacaaagt ggcgaacgtg gtgaccctgg gtggtgcaaa ccgcttaacc
     2041 acaggtgatg cgctgccggg taccgatccg aaccagaaaa ttctgtatac cagcatttac
     2101 agcagcgcgg atgatattgt gatgaactgt ctgagccgcc tggatggcgc gcgcaacgtt
     2161 cagattcatg gcgtgggtca tatgggcctg ctgtatagca gccaggtgaa cagcctgatt
     2221 aaagaaggcc tgaacggtgg cggccagaac accaacggtg gcggttctgg cggtggcggt
     2281 agtggcagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     2341 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     2401 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     2461 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     2521 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     2581 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     2641 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     2701 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     2761 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     2821 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     2881 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     2941 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaaataa
     3001 tgattcccgc tgatagtgct agtgtagatc gctactagta gcggccgctg cagtccggca
     3061 aaaaagggca aggtgtcacc accctgccct ttttctttaa aaccgaaaag attacttcgc
     3121 gttatgcagg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     3181 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     3241 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     3301 gcgtttttcc acaggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     3361 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     3421 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     3481 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     3541 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     3601 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgcaact ggcagcagcc
     3661 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     3721 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     3781 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     3841 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaa
//
