LOCUS       sequence-112180-        6625 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Venus-LUMP fusion with thrombin cleavage 
            site, a FRET-based thrombin sensor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6625)
  AUTHORS   Hoepker AC, Wang A, Le Marois A, Suhling K, Yan Y, Marriott G
  TITLE     Genetically encoded sensors of protein hydrodynamics and molecular 
            proximity.
  JOURNAL   Proc Natl Acad Sci U S A. 2015 Apr 30. pii: 201424021.
  PUBMED    25931526
REFERENCE   2  (bases 1 to 6625)
  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: "Proc Natl 
            Acad Sci U S A. 2015 Apr 30. pii: 201424021."
COMMENT     Sequence Label: 274_pSKB3-Venus-LUMP
FEATURES             Location/Qualifiers
     source          1..6625
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     61..82
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(312..1127)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(312..1127)
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1035..1054
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1119..1138
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1249..1837
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1738..1757
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1991..2008
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2023..2165
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2091..2110)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2251..2273
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2267..2458)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3267..4349)
                     /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     4311..4330
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4350..4427)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4633..4652
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4736..4755
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4736..4754
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4755..4779
                     /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)."
     RBS             4794..4816
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4835..4852
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4874..4894
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(4898..4900,4901..4903,4904..5614)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4943..4964)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5204..5223)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5551..5572
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5621..5638
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             6221..6238
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6291..6309)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6305..6352
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      join(6389..6625,1..219)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6476..6495)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
       61 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
      121 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
      181 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc aggtggcact tttcggggaa
      241 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
      301 tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
      361 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
      421 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
      481 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
      541 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
      601 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
      661 attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
      721 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
      781 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
      841 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
      901 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
      961 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     1021 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     1081 tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta
     1141 ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt
     1201 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     1261 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     1321 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     1381 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     1441 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     1501 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     1561 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     1621 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     1681 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     1741 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     1801 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     1861 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     1921 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     1981 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     2041 ccttacgcat ctgtgcggta tttcacaccg catatatggt gcactctcag tacaatctgc
     2101 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     2161 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     2221 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     2281 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     2341 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     2401 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     2461 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     2521 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     2581 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     2641 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     2701 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     2761 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     2821 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     2881 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
     2941 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     3001 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     3061 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     3121 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     3181 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     3241 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3301 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     3361 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     3421 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     3481 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     3541 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     3601 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     3661 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     3721 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     3781 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     3841 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     3901 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     3961 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     4021 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     4081 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     4141 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     4201 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     4261 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     4321 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     4381 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     4441 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     4501 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     4561 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     4621 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     4681 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata
     4741 cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt
     4801 aactttaaga aggagatata ccatgggcag cagccatcat catcatcatc acgattacga
     4861 tatcccaacg accgaaaacc tttacttcca gggccatatg gtgagcaagg gcgaggagct
     4921 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     4981 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     5041 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct tcggctacgg
     5101 cctgatgtgc ttcgcccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     5161 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     5221 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     5281 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     5341 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     5401 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     5461 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagctacc agtccgccct
     5521 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     5581 cgggatcact ctcggcatgg acgagctata caaggctagc ctggttccgc gtggatcccg
     5641 tggaagcttt agaggtattg ttcaaggtcg tggtgttatt cgatccattt caaaaagtga
     5701 agatagtcag cgacatggga ttgcattccc tgagggtatg tttcaacttg ttgatgtaga
     5761 tactgtaatg ttagtaaatg gttgttcatt gacagttgtc cgtattttag gcgatatggt
     5821 gtactttgat atcgaccaag cattgggtac gacaacattt gatggattaa aagaaggcga
     5881 tcaggtaaac ctagagatcc accctaaatt tggtgaagtg gttggccgtg gtggattaac
     5941 aggaaatatt aaaggcactg cgctggtcgc agcaatagaa gagaatgacg caggtttttc
     6001 tgtactgatt gatatcccta aagggttagc tgaaaactta acagtgaaag atgatattgg
     6061 aattgatggg atctctttgc ctatcactga tatgtcagac agcattatta cattgaatta
     6121 ctcgcgtgat ttgttagcga gtactaacat tgcatcgtta gcaaaagatg tgaaagtgaa
     6181 cgttgaaatt ttaaatgaat ggtaggcggc cgcactcgag caccaccacc accaccactg
     6241 agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     6301 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     6361 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     6421 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     6481 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     6541 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     6601 gattagggtg atggttcacg tagtg
//
