LOCUS       sequence-183562-        6203 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of BtuF which is the vitamin B12 binding protein for 
            BtuCD ABC Transporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6203)
  AUTHORS   Borths EL, Locher KP, Lee AT, Rees DC
  TITLE     The structure of Escherichia coli BtuF and binding to its cognate 
            ATP binding cassette transporter.
  JOURNAL   Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):16642-7. Epub 2002 Dec 
            10.
  PUBMED    12475936
REFERENCE   2  (bases 1 to 6203)
  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."; date: "2002-12-24"; volume: "99(26)"; pages: 
            "16642-7. Epub 2002 Dec 10"
COMMENT     Sequence Label: BtuF
FEATURES             Location/Qualifiers
     source          1..6203
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      10..598
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     499..518
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     752..769
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    784..923
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(849..868)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1009..1031
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1025..1216)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2025..3107)
                     /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     3069..3088
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3108..3185)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3391..3410
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3494..3513
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3494..3512
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3513..3537
                     /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             3552..3574
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     sig_peptide     3582..3647
                     /codon_start=1
                     /gene="pelB (fragment)"
                     /product="leader peptide for secretion"
                     /label=pelB signal sequence
                     /translation="MKYLLPTAAAGLLLLAAQPAMA"
     CDS             4428..4445
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4498..4516)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4512..4559
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4596..5051
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4683..4702)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4893..4914
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5077..5181
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5182..5250,5251..6042)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5400..5419)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
       61 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
      121 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
      181 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
      241 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
      301 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
      361 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
      421 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
      481 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
      541 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
      601 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
      661 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
      721 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      781 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
      841 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
      901 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
      961 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     1021 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     1081 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     1141 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     1201 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     1261 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     1321 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     1381 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     1441 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     1501 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     1561 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     1621 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
     1681 gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
     1741 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
     1801 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
     1861 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     1921 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     1981 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     2041 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     2101 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     2161 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     2221 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     2281 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
     2341 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     2401 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     2461 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     2521 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     2581 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     2641 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     2701 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     2761 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     2821 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     2881 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     2941 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     3001 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     3061 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     3121 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     3181 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     3241 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     3301 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     3361 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     3421 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
     3481 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
     3541 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgaaatac ctgctgccga
     3601 ccgctgctgc tggtctgctg ctcctcgctg cccagccggc gatggccatg gatatcggaa
     3661 ttaattcgga tccggcgccg cgcgtcatca cgctttctcc cgccaacact gaacttgcct
     3721 ttgccgccgg gatcacgccg gttggggtca gcagctattc cgactatcct ccacaagcgc
     3781 aaaagattga gcaggtttcc acctggcagg ggatgaatct ggaacgcatt gtcgcgctga
     3841 aacccgatct ggtgattgcc tggcgtggag gtaatgccga gcggcaggtt gaccagctgg
     3901 cttcgctggg aataaaagtg atgtgggtcg atgcgacaag cattgaacaa attgccaatg
     3961 cgttacgtca actggccccc tggagtccgc aaccagacaa ggccgaacaa gccgcgcaat
     4021 ccctgctgga tcagtacgcg caattgaaag cgcaatatgc tgataaacct aaaaaacgtg
     4081 tttttctgca attcggcatt aatccgccat ttaccagtgg aaaagagtcg attcagaacc
     4141 aggtactcga agtttgtggc ggagaaaaca tctttaaaga cagccgggtt ccctggccgc
     4201 aagttagccg cgaacaggtg ttagcacgct cgccacaggc gattgtcatt acaggcggac
     4261 cggaccaaat tcctaaaatc aaacaatact ggggtgaaca gctcaaaatt cccgttattc
     4321 ctctcacgag tgactggttt gaacgtgcaa gcccacgtat tatcctcgct gcacaacagc
     4381 tctgtaatgc gctttcacag gtagataagc ttgcggccgc actcgagcac caccaccacc
     4441 accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
     4501 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     4561 tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag
     4621 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     4681 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     4741 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     4801 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     4861 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     4921 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     4981 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     5041 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     5101 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     5161 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     5221 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     5281 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     5341 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     5401 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     5461 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     5521 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     5581 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     5641 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     5701 gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact
     5761 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     5821 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     5881 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     5941 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     6001 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     6061 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     6121 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     6181 tcagaccccg tagaaaagat caa
//
