LOCUS       sequence-430077-        6078 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses truncated RvLEAM protein under IPTG induction in bacterial
            host cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6078)
  AUTHORS   Abe KM, Lim CJ
  TITLE     Small LEA proteins as an effective air-water interface protectant 
            for fragile samples during cryo-EM grid plunge freezing.
  JOURNAL   bioRxiv [Preprint]. 2024 Feb 11:2024.02.06.579238. doi: 
            10.1101/2024.02.06.579238.
  PUBMED    38370693
REFERENCE   2  (bases 1 to 6078)
  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: "bioRxiv 
            [Preprint]. 2024 Feb 11:2024.02.06.579238. doi: 
            10.1101/2024.02.06.579238."
COMMENT     Sequence Label: pET15b-RvLEAMshort
FEATURES             Location/Qualifiers
     source          1..6078
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     701..718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    733..872
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(798..817)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     958..980
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(974..1165)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2477..3559)
                     /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     3521..3540
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3560..3637)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3843..3862
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3946..3965
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3946..3964
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3965..3989
                     /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             4004..4026
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4045..4062
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4072..4095
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(4524..4542)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4538..4585
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(4760..4765,4766..4782,4783..4788))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(4815..4833)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4901..5005
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5006..5074,5075..5866)
                     /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(5224..5243)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6037..6078,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      661 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      721 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
      781 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
      841 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
      901 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
      961 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     1021 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     1081 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     1141 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     1201 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     1261 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     1321 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     1381 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     1441 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     1501 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     1561 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     1621 tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga
     1681 tggcggacgc gatggatatg ttctgccaag ggttggtttg cgcattcaca gttctccgca
     1741 agaattgatt ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc
     1801 cattcaggtc gaggtggccc ggctccatgc accgcgacgc aacgcgggga ggcagacaag
     1861 gtatagggcg gcgcctacaa tccatgccaa cccgttccat gtgctcgccg aggcggcata
     1921 aatcgccgtg acgatcagcg gtccaatgat cgaagttagg ctggtaagag ccgcgagcga
     1981 tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc
     2041 gggcatcccg atgccgccgg aagcgagaag aatcataatg gggaaggcca tccagcctcg
     2101 cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg cgataatggc
     2161 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     2221 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     2281 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     2341 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     2401 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     2461 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     2521 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     2581 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     2641 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     2701 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat
     2761 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     2821 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     2881 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     2941 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     3001 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     3061 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     3121 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     3181 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     3241 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     3301 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     3361 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     3421 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     3481 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     3541 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     3601 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     3661 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     3721 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     3781 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     3841 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     3901 acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta
     3961 taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga
     4021 aggagatata ccatgggcag cagccatcat catcatcatc acagcagcgg cctggaagtt
     4081 ctgttccagg ggcccgggaa ggatctcaag aatgaagcct cgtggaaagc caaaggtgtc
     4141 gctaaccaag ccgctggagc attcgaacga gcgaaggata cagtgaagga aggtgtccat
     4201 gacatgaagc gcagtggaag ccgcgtcttc gaacaaggac aggaggaagt agaggctggt
     4261 gctcaacacg caaaggctgg ctatcagagt gccaagaatg ctgcgcaaga cactgccgca
     4321 actcttaaag acaaagccgg cagcgcctgg aatcaagcga aacacgttgt ggaagataag
     4381 ggagaggatg ttgttgaagc agtcaaggac actgcttcaa aagtctgggg caaagccaaa
     4441 catgtggccg aggacgttaa ggagaacgcg tgaggatccg gctgctaaca aagcccgaaa
     4501 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     4561 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat atcccgcaag
     4621 aggcccggca gtaccggcat aaccaagcct atgcctacag catccagggt gacggtgccg
     4681 aggatgacga tgagcgcatt gttagatttc atacacggtg cctgactgcg ttagcaattt
     4741 aactgtgata aactaccgca ttaaagctta tcgatgataa gctgtcaaac atgagaattc
     4801 ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     4861 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     4921 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     4981 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     5041 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     5101 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     5161 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5221 tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg
     5281 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     5341 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     5401 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     5461 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     5521 aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt
     5581 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     5641 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     5701 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     5761 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     5821 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     5881 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     5941 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6001 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6061 aatctgctgc ttgcaaac
//
