LOCUS       sequence-430568-        7458 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for expression of human strep-tagged UCP1 protein in BacMam system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7458)
  AUTHORS   Kang Y, Chen L
  TITLE     Structural basis for the binding of DNP and purine nucleotides onto 
            UCP1.
  JOURNAL   Nature. 2023 Aug;620(7972):226-231. doi: 10.1038/s41586-023-06332-w.
            Epub 2023 Jun 19.
  PUBMED    37336486
REFERENCE   2  (bases 1 to 7458)
  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"; doi: 
            "10.1038/s41586-023-06332-w"; journalName: "Nature"; date: 
            "2023-08"; volume: "620"; issue: "7972"; pages: "226-231"
COMMENT     Sequence Label: pBM-strep-UCP1
FEATURES             Location/Qualifiers
     source          1..7458
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      2..456
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(89..108)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     298..319
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        482..586
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(587..655,656..1447)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(805..824)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1618..2206
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2107..2126
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2360..2377
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  2511..2735
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(2802..3335)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3505..3510,3511..3527,3528..3533))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     promoter        3906..3997
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     3922..3939
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     enhancer        4170..4549
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4550..4753
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4704..4724
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4750..4774
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          4889..5021
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     5004..5023
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             5064..5102
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     CDS             5115..5138
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             5148..5171
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5181..5204
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             5205..5228
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             6159..6176
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    6183..6771
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6236..6256)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6654..6665)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    6898..7032
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6935..6954)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6989..7008
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  7111..7276
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
ORIGIN
        1 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
       61 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
      121 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
      181 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
      241 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt cttaatagtg
      301 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
      361 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
      421 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
      481 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
      541 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
      601 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
      661 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
      721 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
      781 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
      841 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
      901 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
      961 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     1021 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     1081 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     1141 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     1201 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     1261 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     1321 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     1381 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     1441 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     1501 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     1561 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     1621 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     1681 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     1741 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     1801 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     1861 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     1921 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     1981 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     2041 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     2101 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     2161 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     2221 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     2281 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     2341 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     2401 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atagaccagc cgcgtaacct
     2461 ggcaaaatcg gttacggttg agtaataaat ggatgccctg cgtaagcggg tgtgggcgga
     2521 caataaagtc ttaaactgaa caaaatagat ctaaactatg acaataaagt cttaaactag
     2581 acagaatagt tgtaaactga aatcagtcca gttatgctgt gaaaaagcat actggacttt
     2641 tgttatggct aaagcaaact cttcattttc tgaagtgcaa attgcccgtc gtattaaaga
     2701 ggggcgtggc caagggcatg gtaaagacta tattcgcggc gttgtgacaa tttaccgaac
     2761 aactccgcgg ccgggaagcc gatctcggct tgaacgaatt gttaggtggc ggtacttggg
     2821 tcgatatcaa agtgcatcac ttcttcccgt atgcccaact ttgtatagag agccactgcg
     2881 ggatcgtcac cgtaatctgc ttgcacgtag atcacataag caccaagcgc gttggcctca
     2941 tgcttgagga gattgatgag cgcggtggca atgccctgcc tccggtgctc gccggagact
     3001 gcgagatcat agatatagat ctcactacgc ggctgctcaa acttgggcag aacgtaagcc
     3061 gcgagagcgc caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta
     3121 cggagcaagt tcccgaggta atcggagtcc ggctgatgtt gggagtaggt ggctacgtct
     3181 ccgaactcac gaccgaaaag atcaagagca gcccgcatgg atttgacttg gtcagggccg
     3241 agcctacatg tgcgaatgat gcccatactt gagccaccta actttgtttt agggcgactg
     3301 ccctgctgcg taacatcgtt gctgctccat aacatcaaac atcgacccac ggcgtaacgc
     3361 gcttgctgct tggatgcccg aggcatagac tgtacaaaaa aacagtcata acaagccatg
     3421 aaaaccgcca ctgcgccgtt accaccgctg cgttcggtca aggttctgga ccagttgcgt
     3481 gagcgcatac gctacttgca ttacagttta cgaaccgaac aggcttatgt caactgggtt
     3541 cgtgccttca tccgtttcca cggtgtgcgt cacccggcaa ccttgggcag cagcgaagtc
     3601 gaggcatttc tgtcctggct ggcgaacgag cgcaaggttt cggtctccac gcatcgtcag
     3661 gcattggcgg ccttgctgtt cttctacggc aaggtgctgt gcacggatct gccctggctt
     3721 caggagatcg gaagacctcg gccgtcgcgg cgcttgccgg tggtgctgac cccggatgaa
     3781 gtggttcgca tcctcggttt tctggaaggc gagcatcgtt tgttcgccca ggactctagc
     3841 tatagttcta gtggttggct acgtacttaa ttaacaacac catttgtact ccggaatatt
     3901 aatagatcat ggagataatt aaaatgataa ccatctcgca aataaataag tattttactg
     3961 ttttcgtaac agttttgtaa taaaaaaacc tataaatatt ccggattatt cataccgtcc
     4021 caccatcggg cgccaatatt ggccattagc catattattc attggttata tagcataaat
     4081 caatattggc tattggccat tgcatacgtt gtatctatat cataatatgt acatttatat
     4141 tggctcatgt ccaatatgac cgccatgttg gcattgatta ttgactagtt attaatagta
     4201 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     4261 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     4321 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     4381 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagtc cgccccctat
     4441 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttacggga
     4501 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
     4561 ttggcagtac accaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
     4621 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     4681 tcgtaataac cccgccccgt tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     4741 tataagcaga gctcgtttag tgaaccgtca gatcactaga agctttattg cggtagttta
     4801 tcacagttaa attgctaacg cagtcagtgc ttctgacaca acagtctcga acttaagctg
     4861 cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag gttacaagac aggtttaagg
     4921 agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct gataggcacc
     4981 tattggtctt actgacatcc actttgcctt tctctccaca ggtgtccact cccagttcaa
     5041 ttacagctct taaggaattc atgtctcggc tcgaggaaga actgcgccga agactcactg
     5101 agggtagtgg aggttggtct catccacaat ttgagaaagg tagtggagac tacaaggacg
     5161 atgatgacaa gggatcgggg tggagccacc cgcagttcga aaagctcgaa gtgctgttcc
     5221 aaggccctat ggggggcctg acagcctcgg acgtacaccc gaccctgggg gtccagctct
     5281 tctcagctgg aatagcggcg tgcttggcgg acgtgatcac cttcccgctg gacacggcca
     5341 aagtccggct ccaggtccaa ggtgaatgcc cgacgtccag tgttattagg tataaaggtg
     5401 tcctgggaac aatcaccgct gtggtaaaaa cagaagggcg gatgaaactc tacagcgggc
     5461 tgcctgcggg gcttcagcgg caaatcagct ccgcctctct caggatcggc ctctacgaca
     5521 cggtccagga gttcctcacc gcagggaaag aaacagcacc tagtttagga agcaagattt
     5581 tagctggtct aacgactgga ggagtggcag tattcattgg gcaacccaca gaggtcgtga
     5641 aagtcagact tcaagcacag agccatctcc acggaatcaa acctcgctac acggggactt
     5701 ataatgcgta cagaataata gcaacaaccg aaggcttgac gggtctttgg aaagggacta
     5761 ctcccaatct gatgagaagt gtcatcatca attgtacaga gctagtaaca tatgatctaa
     5821 tgaaggaggc ctttgtgaaa aacaacatat tagcagatga cgtcccctgc cacttggtgt
     5881 cggctcttat cgctggattt tgcgcaacag ctatgtcctc cccggtggat gtagtaaaaa
     5941 ccagatttat taattctcca ccaggacagt acaaaagtgt gcccaactgt gcaatgaaag
     6001 tgttcactaa cgaaggacca acggctttct tcaaggggtt ggtaccttcc ttcttgcgac
     6061 ttggatcctg gaacgtcatt atgtttgtgt gctttgaaca actgaaacga gaactgtcaa
     6121 agtcaaggca gactatggac tgtgccacat aactcgagca ccaccaccac caccactgag
     6181 ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     6241 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     6301 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     6361 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     6421 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     6481 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     6541 tgttgggcac tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc
     6601 tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     6661 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     6721 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg cctggtcgag
     6781 aagtactaga ggatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     6841 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     6901 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     6961 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     7021 catgtctgga tctgatcagt aacaacacca tttaaatgga gtggttacaa atggagtggt
     7081 taattaagct gcttgagcct aggagatccg aaccagataa gtgaaatcta gttccaaact
     7141 attttgtcat ttttaatttt cgtattagct tacgacgcta cacccagttc ccatctattt
     7201 tgtcactctt ccctaaataa tccttaaaaa ctccatttcc acccctccca gttcccaact
     7261 attttgtccg cccacagcgg ggcatttttc ttcctgttat gtttttaatc aaacatcctg
     7321 ccaactccat gtgacaaacc gtcatcttcg gctacttttt ctctgtcaca gaatgaaaat
     7381 ttttctgtca tctcttcgtt attaatgttt gtaattgact gaatatcaac gcttatttgc
     7441 agcctgaatg gcgaatgg
//
