LOCUS       sequence-193675-        4042 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for the thermostable anaerobic fluorescent 
            protein tLOV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4042)
  AUTHORS   Higgins SA, Ouonkap SVY, Savage DF
  TITLE     Rapid and Programmable Protein Mutagenesis Using Plasmid 
            Recombineering.
  JOURNAL   ACS Synth Biol. 2017 Jul 24. doi: 10.1021/acssynbio.7b00112.
  PUBMED    28707884
REFERENCE   2  (bases 1 to 4042)
  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: "ACS Synth 
            Biol. 2017 Jul 24. doi: 10.1021/acssynbio.7b00112."
COMMENT     Sequence Label: pTKEI-tLOV
FEATURES             Location/Qualifiers
     source          1..4042
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     154..171
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(379..1461)
                     /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     1423..1442
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1462..1539)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(1584..1589,1590..1606,1607..1613)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1621..1637
                     /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)."
     CDS             2001..2018
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2036..2053)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2206..2292
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2384..2411
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(2517..3332)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSGPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3240..3259
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3324..3343
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3454..4042
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3943..3962
                     /label=pBR322ori-F
                     /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 gagcgccacg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
      241 ctcaacgaca ggagcacgat catgctagtc atgccccgcg cccaccggaa ggagctgact
      301 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
      361 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
      421 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
      481 ttttcttttc accagtgaca cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
      541 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
      601 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
      661 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
      721 atcgttggca accagcatcg cagtgggaac gataccctca ttcagcattt gcatggtttg
      781 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
      841 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
      901 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
      961 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     1021 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     1081 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     1141 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     1201 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     1261 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     1321 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     1381 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     1441 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     1501 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     1561 tatgcgactc cgatcccgca aatttgacaa ttaatcatcc ggctcgtata atgtgtggaa
     1621 ttgtgagcgg ataacaattt cacacaagga gatataccat gtccatcgaa aaaaacttcg
     1681 ttatcaccga cccgagctta cctgacaatc caatcatctt tgcctcagat ggttttctcg
     1741 agctaaccga atacagccgt gaagaagttc tgggtcgtaa tgctcgtttc ctgcagggcc
     1801 cggaaaccga ccaagctacg gttcagaaaa ttcgcgatgc aattcgcgat cagcgtgaga
     1861 ctaccgttca attgatcaac tacaccaaaa gcggtaaaaa attttggaac ctgttgcatc
     1921 ttcaaccggt atttgaccaa aaaggcgagc ttcagtactt tatcggcgtg cagctggatg
     1981 ttagtgacca tgtgagcggt caccatcacc atcaccatta agagaagatt ttcagcctga
     2041 tacagattaa atcagaacgc agaagcggtc tgataaaaca gaatttgcct ggcggcagta
     2101 gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt agcgccgatg
     2161 gtagtgtggg gtatccccat gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag
     2221 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     2281 agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg
     2341 cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc catcctgacg
     2401 gatggccttt tctgaaagga ggaactatat ccggatatcc acaggacggg tgtggtcgcc
     2461 atgatcgcgt agtcaaggtt tcttagacgt caggtggcac ttttcgggga aatgtgttag
     2521 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     2581 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     2641 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     2701 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     2761 tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca
     2821 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     2881 tgagcgaggc gaaatacgcg atcactgtta aaaggacaat tacaaacagg aatcgaatgc
     2941 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     3001 tctagtacct ggaatgctgt tttgccgggg atcgcagtgg tgagtaacca tgcatcatca
     3061 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     3121 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     3181 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
     3241 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccca
     3301 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
     3361 gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     3421 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     3481 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     3541 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     3601 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     3661 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     3721 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     3781 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     3841 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     3901 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     3961 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     4021 aggggggcgg agcctatgga aa
//
