LOCUS       sequence-435554-        5740 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The pSBLego vector enables sybodies for the Legobody method with a 
            PelB signal for periplasmic expression and a ""SLEHHHHHH"" motif for
            purification and Legobody Fab binding..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5740)
  AUTHORS   Ackle F, Thavarasah S, Earp JC, Seeger MA
  TITLE     Rigid enlargement of sybodies with antibody fragments for cryo-EM 
            analyses of small membrane proteins.
  JOURNAL   Sci Rep. 2025 Mar 19;15(1):9460. doi: 10.1038/s41598-025-92950-5.
  PUBMED    40108246
REFERENCE   2  (bases 1 to 5740)
  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: "Sci Rep."; 
            date: "2025-03-19"; volume: "15(1):9460. doi"; pages: " 
            10.1038/s41598-025-92950-5"
COMMENT     Sequence Label: pSBLego
FEATURES             Location/Qualifiers
     source          1..5740
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..915
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(841..860)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1001..1023
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1179..2057)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        2084..2368
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     2291..2310
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     sig_peptide     2402..2467
                     /codon_start=1
                     /gene="pelB (fragment)"
                     /product="leader peptide for secretion"
                     /label=pelB signal sequence
                     /translation="MKYLLPTAAAGLLLLAAQPAMA"
     CDS             complement(2516..2821)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(2618..2637)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(3163..3822)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3737..3756
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(3876..3882,3883..3900,3901..3906))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(3931..4055)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(4074..4094)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(4098..4118)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4151..4168
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4224..4241)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4394..4480
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4572..4599
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        4618..4709
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4710..4778,4779..5570)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4928..4947)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag tgcccctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
      841 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
      901 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
      961 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     1021 aggttttcac cgtcatcacc gaaacgcgcg aggcagcaga tcaattcgcg cgcgaaggcg
     1081 aagcggcatg cataatgtgc ctgtcaaatg gacgaagcag ggattctgca aaccctatgc
     1141 tactccgtca agccgtcaat tgtctgattc gttaccaatt atgacaactt gacggctaca
     1201 tcattcactt tttcttcaca accggcacgg aactcgctcg ggctggcccc ggtgcatttt
     1261 ttaaataccc gcgagaaata gagttgatcg tcaaaaccaa cattgcgacc gacggtggcg
     1321 ataggcatcc gggtggtgct caaaagcagc ttcgcctggc tgatacgttg gtcctcgcgc
     1381 cagcttaaga cgctaatccc taactgctgg cggaaaagat gtgacagacg cgacggcgac
     1441 aagcaaacat gctgtgcgac gctggcgata tcaaaattgc tgtctgccag gtgatcgctg
     1501 atgtactgac aagcctcgcg tacccgatta tccatcggtg gatggagcga ctcgttaatc
     1561 gcttccatgc gccgcagtaa caattgctca agcagattta tcgccagcag ctccgaatag
     1621 cgcccttccc cttgcccggc gttaatgatt tgcccaaaca ggtcgctgaa atgcggctgg
     1681 tgcgcttcat ccgggcgaaa gaaccccgta ttggcaaata ttgacggcca gttaagccat
     1741 tcatgccagt aggcgcgcgg acgaaagtaa acccactggt gataccattc gcgagcctcc
     1801 ggatgacgac cgtagtgatg aatctctcct ggcgggaaca gcaaaatatc acccggtcgg
     1861 caaacaaatt ctcgtccctg atttttcacc accccctgac cgcgaatggt gagattgaga
     1921 atataacctt tcattcccag cggtcggtcg ataaaaaaat cgagataacc gttggcctca
     1981 atcggcgtta aacccgccac cagatgggca ttaaacgagt atcccggcag caggggatca
     2041 ttttgcgctt cagccatact tttcatactc ccgccattca gagaagaaac caattgtcca
     2101 tattgcatca gacattgccg tcactgcgtc ttttactggc acttctcgct aaccaaaccg
     2161 gtaaccccgc ttattaaaag cattctgtaa caaagcggga ccaaagccat gacaaaaacg
     2221 cgtaacaaaa gtgtctataa tcacggcaga aaagtccaca ttgattattt gcacggcgtc
     2281 acactttgct atgccatagc atttttatcc ataagattag cggatcctac ctgacgcttt
     2341 ttatcgcaac tctctactgt ttctccatac ccgttttttg ggctaacagg aggaattaac
     2401 catgaaatac ctgctgccga ccgctgctgc cggactgctg ctcctcgctg cccagcctgc
     2461 aatggccagt tgaagagcga cctgcagact ggctgtgtat aagggagcct gacatttata
     2521 ttccccagaa catcaggtta atggcgtttt tgatgtcatt ttcgcggtgg ctgagatcag
     2581 ccacttcttc cccgataacg gagaccggca cactggccat atcggtggtc atcatgcgcc
     2641 agctttcatc cccgatatgc accaccgggt aaagttcacg ggagacttta tctgacagca
     2701 gacgtgcact ggccaggggg atcaccatcc gtcgcccggg cgtgtcaata atatcactct
     2761 gtacatccac aaacagacga taacggctct ctcttttata ggtgtaaacc ttaaactgca
     2821 tttcaccagc ccctgttctc gtcagcaaaa gagccgttca tttcaataaa ccgggcgacc
     2881 tcagccatcc cttcctgatt ttccgctttc cagcgttcgg cacgcagacg acgggcttca
     2941 ttctgcatgg ttgtgcttac cagaccggag atattgacat catatatgcc ttgagcaact
     3001 gatagctgtc gctgtcaact gtcactgtaa tacgctgctt catagcatac ctctttttga
     3061 catacttcgg gtatacatat cagtatatat tcttataccg caaaaatcag cgcgcaaata
     3121 cgcatactgt tatctggctt ttagtaagcc ggatccacgc gtttacgccc cgccctgcca
     3181 ctcatcgcag tactgttgta attcattaag cattctgccg acatggaagc catcacaaac
     3241 ggcatgatga acctgaatcg ccagcggcat cagcaccttg tcgccttgcg tataatattt
     3301 gcccatggtg aaaacggggg cgaagaagtt gtccatattg gccacgttta aatcaaaact
     3361 ggtgaaactc acccagggat tggctgagac gaaaaacata ttctcaataa accctttagg
     3421 gaaataggcc aggttttcac cgtaacacgc cacatcttgc gaatatatgt gtagaaactg
     3481 ccggaaatcg tcgtggtatt cactccagag cgatgaaaac gtttcagttt gctcatggaa
     3541 aacggtgtaa caagggtgaa cactatccca tatcaccagc tcaccgtctt tcattgccat
     3601 acggaattcc ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa
     3661 cttgtgctta tttttcttta cggtctttaa aaaggccgta atatccagct gaacggtctg
     3721 gttataggta cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac gatgccattg
     3781 ggatatatca acggtggtat atccagtgat ttttttctcc attttagctt ccttagctcc
     3841 tgaaaatctc gacggatcct aactcaaaat ccacacatta tacgagccgg aagcataaag
     3901 tgtaaagcct ggggtgccta atgcggccgc catagtgact ggatatgttg tgttttacag
     3961 tattatgtag tctgtttttt atgcaaaatc taatttaata tattgatatt tatatcattt
     4021 tacgtttctc gttcagcttt tttgtacaaa cttgtgatat cgaattcgct agctaccttt
     4081 ctcttctttt ttggatctac ctttctcttc ttttttgggg atccactagt tcagctcttc
     4141 ttccttggaa caccatcacc accaccattg agtttaaacg gtctccagct tggctgtttt
     4201 ggcggatgag agaagatttt cagcctgata cagattaaat cagaacgcag aagcggtctg
     4261 ataaaacaga atttgcctgg cggcagtagc gcggtggtcc cacctgaccc catgccgaac
     4321 tcagaagtga aacgccgtag cgccgatggt agtgtggggt ctccccatgc gagagtaggg
     4381 aactgccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat
     4441 ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa
     4501 cgttgcgaag caacggcccg gagggtggcg ggcaggacgc ccgccataaa ctgccaggca
     4561 tcaaattaag cagaaggcca tcctgacgga tggccttttt gcgtttctac aaactctttt
     4621 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     4681 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     4741 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     4801 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     4861 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     4921 aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc
     4981 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     5041 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     5101 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     5161 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     5221 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     5281 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     5341 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     5401 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     5461 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     5521 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     5581 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     5641 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     5701 actgagcgtc agaccccgta gaaaagatca aaggatcttc
//
