LOCUS       sequence-178594-        7204 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of BtuCD in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7204)
  AUTHORS   Locher KP, Lee AT, Rees DC
  TITLE     The E. coli BtuCD structure: a framework for ABC transporter 
            architecture and mechanism.
  JOURNAL   Science. 2002 May 10;296(5570):1091-8.
  PUBMED    12004122
REFERENCE   2  (bases 1 to 7204)
  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: "Science."; 
            date: "2002-05-10"; volume: "296(5570)"; pages: "1091-8"
COMMENT     Sequence Label: BtuCD
FEATURES             Location/Qualifiers
     source          1..7204
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(948..2030)
                     /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     1992..2011
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2031..2108)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2314..2333
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2417..2436
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2417..2435
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2436..2460
                     /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             2475..2497
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2510..2539
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2555..2569
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     RBS             3566..3588
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(4419..4437)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4433..4480
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4517..4972
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4604..4623)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4814..4835
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4998..5102
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5103..5171,5172..5963)
                     /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(5321..5340)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6134..6722
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6623..6642
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6876..6893
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6908..7050
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6976..6995)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7136..7158
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(7152..7204,1..139))
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
       61 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
      121 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
      181 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
      241 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
      301 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
      361 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
      421 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
      481 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
      541 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
      601 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
      661 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
      721 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
      781 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
      841 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
      901 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
      961 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     1021 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     1081 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     1141 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     1201 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta
     1261 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     1321 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     1381 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     1441 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     1501 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     1561 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     1621 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     1681 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     1741 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     1801 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     1861 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     1921 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     1981 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     2041 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     2101 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
     2161 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc
     2221 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
     2281 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
     2341 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
     2401 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
     2461 cctctagaaa taattttgtt taactttaag aaggagatat accatgggcc atcatcatca
     2521 tcatcatcat catcatcaca gcagcggcca tatcgacgac gacgacaagc atatgctgac
     2581 acttgcccgc caacaacagc gacaaaatat tcgctggtta ttatgcctgt cagttttgat
     2641 gctgctggcg cttctcttaa gcctttgcgc cggtgaacaa tggatctcgc caggtgactg
     2701 gtttactcct cgtggcgaac tgttcgtctg gcaaattcgc ctgccacgta cgctggctgt
     2761 attgctggtt ggtgcggcgc tggctatatc cggcgctgta atgcaggcgt tgtttgaaaa
     2821 tcctctggca gaacctggac tacttggcgt ctctaacggc gcaggcgtgg ggcttatcgc
     2881 cgcggtattg cttgggcaag ggcaactccc caactgggcg ctagggctgt gtgcgattgc
     2941 tggcgcgctt atcatcactt taatactctt acgtttcgcc cgtcgtcatc tttcgaccag
     3001 tcggttattg ctggctggcg ttgcattagg gattatctgt agcgcactaa tgacgtgggc
     3061 tatctacttt tccacctcag ttgatttgcg tcagctgatg tactggatga tgggcggttt
     3121 tggcggcgta gactggcggc aaagctggct gatgctggca ttgatccccg tgttgttgtg
     3181 gatctgttgt cagtccaggc cgatgaatat gttagcactt ggcgagatct cggcgcggca
     3241 actgggttta cccctgtggt tctggcgcaa tgtgctggtg gcagcgaccg gctggatggt
     3301 tggcgtcagt gtggcgctgg cgggtgctat cggctttatt ggtctggtga tcccccatat
     3361 tctccggttg tgtggtttaa ccgatcatcg cgtattactt cccggctgcg cgctggcagg
     3421 ggcgagcgca ttgctgctgg ccgatattgt agcgcgcctg gcattagctg ccgcagagct
     3481 gcctattggc gtggtcaccg caacgttagg tgcgccggtg tttatctggt tattgttaaa
     3541 agcaggacgt tagtctagaa ataattttgt ttaactttaa gaaggagata tacatatgtc
     3601 tattgtgatg cagttacaag atgttgcgga atctacccgc ctggggccgc tttctggcga
     3661 ggttcgggct ggggagatcc tgcacctggt ggggccgaat ggcgcgggta agagtacctt
     3721 actggcgcga atggccggaa tgaccagcgg taagggaagc attcagttcg cggggcaacc
     3781 actggaagca tggtccgcaa caaaactcgc gctgcatcgc gcctatcttt cacaacagca
     3841 gacgccgccg tttgcaacgc cggtctggca ctacctgaca ctgcatcagc acgataaaac
     3901 gcgtaccgaa ctactgaatg atgtcgcagg ggcgctggct cttgatgaca aactcggacg
     3961 tagcaccaat caactttccg gcggtgaatg gcaacgcgta cgtcttgctg cggtggtgtt
     4021 gcaaatcaca ccacaagcca atcccgcagg ccaattgctg cttcttgatg agccgatgaa
     4081 cagtcttgat gttgcgcaac aaagtgcgtt agacaaaatt ctgagcgcgc tgtgtcagca
     4141 aggactggcg attgtgatga gcagtcacga tctcaaccac acattgcgtc atgcgcatcg
     4201 ggcgtggttg ctaaaaggtg gaaaaatgct ggccagtgga cgcagggaag aggtgctcac
     4261 gccgccaaat ctggcgcagg cctatgggat gaattttcgc cgtctggata tcgaaggtca
     4321 cagaatgctg atttcgacca tcctcgagta gcaccaccac caccactgag atccggctgc
     4381 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
     4441 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
     4501 cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4561 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     4621 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     4681 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     4741 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     4801 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     4861 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     4921 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     4981 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5041 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     5101 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     5161 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     5221 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     5281 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     5341 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     5401 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     5461 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     5521 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     5581 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     5641 tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     5701 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     5761 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     5821 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     5881 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     5941 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     6001 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     6061 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     6121 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     6181 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     6241 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     6301 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     6361 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     6421 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     6481 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     6541 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     6601 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     6661 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     6721 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     6781 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     6841 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     6901 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat
     6961 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     7021 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     7081 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     7141 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     7201 agct
//
