LOCUS       sequence-456035-        3894 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid that encodes a B. Subtilis lipase (BsLipA) gene fusion with 
            N-terminal SNAP and C-Terminal eGFP under a T7 promoter. 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 Triple Emulsion Picoreactors for Screening Reactions 
            in Biphasic Environments
  JOURNAL   Adv Mater Interfaces 2024, 12 (3), 2400403. doi: 
            10.1002/admi.202400403.
REFERENCE   2  (bases 1 to 3894)
  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: "Adv Mater 
            Interfaces 2024, 12 (3), 2400403. doi: 10.1002/admi.202400403."
COMMENT     Sequence Label: SMT503
FEATURES             Location/Qualifiers
     source          1..3894
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             923..1636
                     /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             923..1633
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     primer_bind     complement(962..983)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1223..1242)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1570..1591
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1669..1689
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      1690..1747
                     /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(1771..1788)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1942..2530)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2022..2041)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2713..2807)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(2828..3487)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGIMLNELQQYCDEWQGGA"
     primer_bind     3402..3421
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     3625..3644
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3625..3643
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(3668..3894,1..319)
                     /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"
ORIGIN
        1 tgaagaattt ccggttccgg cgctgcatca tccggtgttt cagcaggaga gctttacccg
       61 tcaggtgctg tggaaactgc tgaaagtggt taaatttggc gaagtgatta gctatcagca
      121 gctggcggcc ctggcgggta atccggctgc caccgccgcc gttaaaaccg cgctgagcgg
      181 taacccggtg ccgattctga ttccgtgcca tcgtgtggtt agctctagcg gtgcggttgg
      241 cggttatgaa ggtggtctgg cggtgaaaga gtggctgctg gcccatgaag gtcatcgtct
      301 gggtaaaccg ggtctgggag gtggagggtc tgggggagga ggctcaggcg cggaacataa
      361 cccggtggtg atggtgcatg gcattggtgg cgcgagcttt aactttgcgg gcattaaatc
      421 ctatctggtg agccagggct ggagccagaa cgatctgtat gcggtggatt tctgggataa
      481 aaccggtacc aactataaca acggcccggt gctgagccgt tttgtgcaga aagtgctgga
      541 cgaaaccggc gccaagaaag tggatatcgt ggcgcatagc atgggcggcg cgaacaccct
      601 gtattatatt aagaacctgg acggcggtaa caaagtggcg aacgtggtga ccttgggtgg
      661 tgcaaaccgt ctgactaccg gtgatgcgct gccgggtacc gatccgaacc agaaaattct
      721 gtataccagc atctatagca gcgcggatga tattgtgatg aactgcctga gccgcctgga
      781 tggcgcgcgt aacgttcaga ttcatggcgt gggtcatatg ggcctgctgt atagcagcca
      841 ggtgaacagc ctgattaaag aaggtttgaa cggcggcggc cagaacacca acggtggcgg
      901 ttctggcggt ggcggtagtg gcagcaaggg cgaggagctg ttcaccgggg tggtgcccat
      961 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     1021 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     1081 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     1141 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     1201 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     1261 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     1321 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     1381 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     1441 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     1501 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     1561 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     1621 cgagctgtac aaataatgat tcccgctgat agtgctagtg tagatcgcta ctagtagcgg
     1681 ccgctgcagt ccggcaaaaa agggcaaggt gtcaccaccc tgcccttttt ctttaaaacc
     1741 gaaaagatta cttcgcgtta tgcaggcttc ctcgctcact gactcgctgc gctcggtcgt
     1801 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     1861 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     1921 aaaggccgcg ttgctggcgt ttttccacag gctccgcccc cctgacgagc atcacaaaaa
     1981 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     2041 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     2101 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     2161 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     2221 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     2281 gcaactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     2341 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     2401 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     2461 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     2521 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     2581 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     2641 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     2701 ctcgaggctt ggattctcac caataaaaaa cgcccggcgg caaccgagcg ttctgaacaa
     2761 atccagatgg agttatgagg tcattactgg atctatcaac aggagtccaa gcgagctcga
     2821 tatcaaatta cgccccgccc tgccactcat cgcagtactg ttgtaattca ttaagcatta
     2881 tgccgacatg gaagccatca caaacggcat gatgaacctg aatcgccagc ggcatcagca
     2941 ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac gggggcgaag aagttgtcca
     3001 tattggccac gtttaaatca aaactggtga aactcaccca gggattggct gagacgaaaa
     3061 acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa cacgccacat
     3121 cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc cagagcgatg
     3181 aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta tcccatatca
     3241 ccagctcacc gtctttcatt gccatacgaa attccggatg agcattcatc aggcgggcaa
     3301 gaatgtgaat aaaggccgga taaaacttgt gcttattttt ctttacggtc tttaaaaagg
     3361 ccgtaatatc cagctgaacg gtctggttat aggtacattg agcaactgac tgaaatgcct
     3421 caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca gtgatttttt
     3481 tctccatttt agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta
     3541 gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgcccgatc aactcgagtg
     3601 ccacctgacg tctaagaaac cgaataatac gactcactat agggcttaag tataaggaga
     3661 tatacatatg gacaaagatt gcgaaatgaa acgtaccacc ctggatagcc cgctgggcaa
     3721 actggaactg agcggctgcg aacagggcct gcatgaaatt aaactgctgg gtaaaggcac
     3781 cagcgcggcc gatgcggttg aagttccggc cccggccgcc gtgctgggtg gtccggaacc
     3841 gctgatgcag gcgaccgcgt ggctgaacgc gtattttcat cagccggaag cgat
//
