LOCUS       Exported                6825 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Fluorescent biosensor for extracellular L-lactate.
ACCESSION   .
VERSION     .
KEYWORDS    pAEMXT-eLACCO1.1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6825)
  AUTHORS   Yusuke Nasu, Ciaran Murphy-Royal, Yurong Wen, Jordan Haidey, M. 
            Rosana S. Molina, Abhi Aggarwal, Shuce Zhang, Yuki Kamijo, Marie-Eve
            Paquet, Kaspar Podgorski, Mikhail Drobizhev, Jaideep S. Bains, M. 
            Joanne Lemieux, Grant R. Gordon, Robert E. Campbell
  TITLE     A genetically encoded fluorescent biosensor for extracellular 
            L-lactate
  JOURNAL   bioRxiv
REFERENCE   2  (bases 1 to 6825)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6825
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        554..572
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     1490..1511
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1617..1638)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1878..1897)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2666..2685)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2667..2685)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    2959..3080
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2996..3015)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3050..3069
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     terminator      3108..3135
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3154..3245
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3246..4106
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEELFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3464..3483)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        4269..4626
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(4292..4312)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4477..4612
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4539..4558
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4661..5455
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4715..4734)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5325..5344
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5643..5662)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5687..5734
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6063..6651
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6552..6571
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgatt
      541 acgccaagct cgaaattaac cctcactaaa gggaacaaaa gctggagctc caccgcggtg
      601 gcggccgcga tatcaccatg ggaatccaag gagggtctgt cctgttcggg ctgctgctcg
      661 tcctggctgt cttctgccat tcaggtcata gcagatctat gttctctccc ttggcagtgg
      721 cccaggctcg acgctatagg tggaggattc aaaccgcgtg ggatgctgga acagtaggct
      781 acagtctgtt ccaaaagttt acggagcggg taaaggaact gacggatgga cagcttgagg
      841 tccagccttt tccagcgggt gcagtcgtag gcacgtttga tatgttcgac gcggtcaaga
      901 ctggagtttt ggacggaatg aatcctttca cgctgttttg ggcagggagg atgccggtta
      961 cagctttcct ctccagctac gccctcgggt tggaccgacc tgaccagtgg gaaacatggt
     1021 tttactccct gggtggcctc gacaacgccc gacgagcctt tgccgagcaa ggcctcttct
     1081 acgtgggacc agttcagcac gacctcaata caattcattc aaggaagccg atccggagat
     1141 ttgaggactt caaaggtgtc aagctccgag tgccgggcgg catgattgcg gaagtttttg
     1201 cggccgcagg agcttccacc gtcctcttgc ctggggggga ggtctacccc gctctggaaa
     1261 gaggggtaat agattggagc cacaacgtct atatcatggc cgacaagcag aggaacggca
     1321 tcaaggcgaa cttcgagatc cgccacaaca tcgaggacgg cggcgtgcag ctcgcctacc
     1381 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     1441 tgagcaccca gaccaaactt tcgaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     1501 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagggcggta
     1561 ccggagggag aatggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     1621 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggtgagggcg
     1681 atgccacctc cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     1741 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     1801 accacatgaa gcagcacgac ttcttcaagt cggccatgcc cgaaggctac atccaggagc
     1861 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     1921 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     1981 tcctggggca taagctggag tacagcttta acgatggtgg tgcggctgat tttgttgggc
     2041 ctgcagtaaa ctataacttg gggttccatc aggtggcaaa gtacatcata atggggcctc
     2101 cagagacgcc tgccattcat caacctgtag acctcatgga cttcaccata aaccttaatc
     2161 ggtggcgcag ccttccaaag cctctgcaag aaagattcat cgcggcggtt cacgagtact
     2221 cttggattca ctacgcaggc attcagaagg ctaatttgga ggcgtggccg aaatatcggc
     2281 aggcaggtgt tgaggtcatc cggctgagca acgaggatgt tagaaaattc aggcgactcg
     2341 ctattcccat ttggtttaag tgggctaaga tggataaata ctcacgagag gcattcgcgt
     2401 cccaactgga gtatatgaaa ggtataggtt acgttacgga cgaggaattg aagggcttga
     2461 gcctgggctc cacctccggc tccggcaagc ccggctccgg cgagggctcc accaagggcg
     2521 aattccttga aaatggtggg acatccttat cagagaaaac agttcttctg ctggtgactc
     2581 catttctggc agcagcctgg agccttcatc cctaaggatc ctgactcgag ggggggcccg
     2641 gtaccaggta agtgtaccca attcgcccta tagtgagtcg tattacaatt cactcgatcg
     2701 cccttcccaa cagttgcgca gcctgaatgg cgaatggaga tccaattttt aagtgtataa
     2761 tgtgttaaac tactgattct aattgtttgt gtattttaga ttcacagtcc caaggctcat
     2821 ttcaggcccc tcagtcctca cagtctgttc atgatcataa tcagccatac cacatttgta
     2881 gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg
     2941 aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     3001 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     3061 aaactcatca atgtatctta acgcgtaaat tgtaagcgtt aataagcaga aggccatcct
     3121 gacggatggt ctttttgcgt ttctacaaac tcttttgttt atttttctaa atacattcaa
     3181 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     3241 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     3301 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     3361 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     3421 gccccgaaga actttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     3481 tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     3541 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     3601 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     3661 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     3721 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     3781 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     3841 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     3901 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     3961 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     4021 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     4081 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     4141 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     4201 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac attgaaaaag
     4261 gaagaatcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
     4321 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     4381 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     4441 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     4501 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     4561 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     4621 tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     4681 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     4741 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     4801 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct
     4861 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     4921 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     4981 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     5041 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     5101 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gaacatcagg ggctcgcgcc
     5161 agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac
     5221 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     5281 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     5341 tattgctgaa gaacttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     5401 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     5461 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     5521 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     5581 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta
     5641 actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga
     5701 cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc
     5761 tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc
     5821 ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg
     5881 gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt
     5941 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6001 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6061 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6121 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6181 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6241 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6301 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6361 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6421 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6481 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6541 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6601 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6661 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     6721 gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcattagtt attaatagta
     6781 atcaattacg gggtcattag ttcatagccc atatatggag ttccg
//
