LOCUS       sequence-218616-        6759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FRB-PafA in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6759)
  AUTHORS   Liu Q, Zheng J, Sun W, Huo Y, Zhang L, Hao P, Wang H, Zhuang M
  TITLE     A proximity-tagging system to identify membrane protein-protein 
            interactions.
  JOURNAL   Nat Methods. 2018 Aug 13. pii: 10.1038/s41592-018-0100-5. doi: 
            10.1038/s41592-018-0100-5.
  PUBMED    30104635
REFERENCE   2  (bases 1 to 6759)
  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: "Nat 
            Methods. 2018 Aug 13. pii: 10.1038/s41592-018-0100-5. doi: 
            10.1038/s41592-018-0100-5."
COMMENT     Sequence Label: pGEX6p-1-FRB-PafA
FEATURES             Location/Qualifiers
     source          1..6759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..654
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     612..634
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             661..684
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             709..978
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     primer_bind     complement(2552..2574)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2719..2737)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2805..2909
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2910..2978,2979..3770)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3128..3147)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3941..4529
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4430..4449
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4683..4700
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4773..4850
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4851..5933
                     /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     complement(4870..4889)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5982..5987,5988..6005,6006..6012)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6020..6036
                     /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)."
     primer_bind     6044..6060
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6044..6060
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             6056..6583
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(6076..6093)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6076..6092)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6085..6107)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(6685..6690,6691..6706,6707..6713)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    6721..6737
                     /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)."
ORIGIN
        1 atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt
       61 ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa
      121 tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat
      181 ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac
      241 atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg
      301 gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt
      361 gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa
      421 acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat
      481 gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa
      541 aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca
      601 tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat
      661 ctggaagttc tgttccaggg gcccctggga tccccgattc tgtggcatga gatgtggcac
      721 gagggactcg aagaggccag caggctgtac tttggcgaga ggaacgtcaa gggcatgttc
      781 gaagtgctgg agcccctcca tgcgatgatg gaaaggggcc cacagaccct gaaggagacc
      841 agcttcaacc aggcttacgg cagggacctg atggaggcac aggaatggtg caggaagtac
      901 atgaagagcg gcaacgtgaa agacctgacc caggcgtggg acctctacta ccacgtgttc
      961 aggaggatca gcaagcagtc cggtggcggc agcggtgggg gcagtggagg cggaagcgga
     1021 gggggctctg gcatgagtac cgtggaatcc gcattgaccc gcaggatcat gggcattgaa
     1081 acggagtatg gcctcacctt tgttgatggt gattccaaaa agcttcgccc agatgagata
     1141 gctcgaagga tgtttcgtcc catcgtggag aaatattcca gctctaatat cttcataccc
     1201 aatggttccc ggttgtatct tgatgtgggt tcccacccgg agtacgccac cgccgagtgt
     1261 gataatttga cccagctgat caattttgaa aaagctggcg atgttattgc agatcgcatg
     1321 gctgtagatg ccgaagagtc gctggcgaaa gaagacattg ctgggcaggt gtacctgttt
     1381 aaaaacaatg tcgattccgt gggcaattct tatggctgcc acgaaaacta ccttgtgggt
     1441 cgctccatgc cgttgaaggc gttgggtaaa aggctgatgc cgtttctgat tacccgccag
     1501 ctcatctgcg gcgccggcag gatccatcac cccaatcctt tggataaagg cgaatccttc
     1561 cccttgggct actgcatatc ccagcgctct gaccacgtgt gggagggcgt atcaagtgcc
     1621 accactagat cacgccccat tatcaacacc cgtgatgagc cacatgcgga ttcccattct
     1681 taccgcaggc tgcacgtgat tgtgggtgat gccaacatgg cagagcccag catcgcgttg
     1741 aaggtcggct ccacgttgct ggttctggaa atgattgagg cagatttcgg tttgcccagc
     1801 ttagagcttg ccaatgatat tgcctcaatt agggaaatct cccgcgatgc aacaggatcc
     1861 acactgttgt ccctgaaaga tggcaccacc atgactgcct tgcagatcca gcaggtggtc
     1921 tttgagcatg cctcgaagtg gttggagcag cgccccgaac cagaattttc tggcacctcc
     1981 aacacagaga tggcccgcgt gctggatctg tggggtcgca tgttgaaagc gattgagtcc
     2041 ggtgatttca gcgaagtgga tacagaaatt gactgggtga tcaaaaagaa gctcattgat
     2101 cgtttcattc agcgcggcaa ccttgggttg gatgatccaa aacttgccca agtggacttg
     2161 acttatcacg atattaggcc aggtagaggc ctatttagcg tgctgcaaag ccgcggcatg
     2221 atcaaacggt ggactactga tgaggcgatt ttagctgcgg tggataccgc tcctgataca
     2281 acacgtgctc atttgcgcgg gcgaatcctt aaagcggcgg atactctggg agtacctgtg
     2341 actgtcgatt ggatgcgtca caaggtcaac cgaccggagc cacaatcggt ggaattgggg
     2401 gatccttttt ccgcagtgaa ttccgaggtt gatcagttga ttgagtatat gaccgttcat
     2461 gcggagagct atcgcagtta agtcgactcg agcggccgca tcgtgactga ctgacgatct
     2521 gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
     2581 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
     2641 gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata
     2701 attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     2761 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     2821 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     2881 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     2941 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     3001 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     3061 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     3121 aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc
     3181 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     3241 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     3301 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     3361 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     3421 taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac
     3481 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     3541 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     3601 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     3661 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     3721 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     3781 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     3841 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     3901 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3961 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     4021 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4081 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4141 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4201 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4261 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4321 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4381 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4441 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4501 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4561 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     4621 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     4681 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     4741 atctgtgcgg tatttcacac cgcataaatt ccgacaccat cgaatggcgc aaaacctttc
     4801 gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag
     4861 taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg
     4921 tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg
     4981 agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga
     5041 ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta
     5101 aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg
     5161 tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca
     5221 ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc
     5281 cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg
     5341 aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc
     5401 tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat
     5461 atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt
     5521 ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg
     5581 ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg
     5641 ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc
     5701 cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct
     5761 tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg
     5821 tgaaaagaaa aaccacgcta gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     5881 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     5941 gcaattaatg taagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     6001 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     6061 catgattacg gattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     6121 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     6181 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgctttgc
     6241 ctggtttccg gcaccagaag cggtgccgga aagctggctg gagtgcgatc ttcctgaggc
     6301 cgatactgtc gtcgtcccct caaactggca gatgcacggt tacgatgcgc ccatctacac
     6361 caacgtgacc tatcccatta cggtcaatcc gccgtttgtt cccacggaga atccgacggg
     6421 ttgttactcg ctcacattta atgttgatga aagctggcta caggaaggcc agacgcgaat
     6481 tatttttgat ggcgttggaa ttagcttatc gactgcacgg tgcaccaatg cttctggcgt
     6541 caggcagcca tcggaagctg tggtatggct gtgcaggtcg taaatcactg cataattcgt
     6601 gtcgctcaag gcgcactccc gttctggata atgttttttg cgccgacatc ataacggttc
     6661 tggcaaatat tctgaaatga gctgttgaca attaatcatc ggctcgtata atgtgtggaa
     6721 ttgtgagcgg ataacaattt cacacaggaa acagtattc
//
