LOCUS       40924_26869        5552 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pKLD66nCBP, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5552)
  AUTHORS   St Maurice M.
  TITLE     The absence of substrate channeling in the biotin-dependent enzyme 
            urea amidolyase
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5552)
  AUTHORS   St Maurice M.
  TITLE     Direct Submission
  JOURNAL   Submitted (04-JUN-2015) Biological Sciences, Marquette University, 
            530 N 15th Street, Milwaukee, WI 53233, USA
REFERENCE   3  (bases 1 to 5552)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5552)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (04-JUN-2015) Biological Sciences, Marquette University, 530 N 15th 
            Street, Milwaukee, WI 53233, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5552
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        368..386
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    387..411
                     /label=lac operator
                     /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             426..448
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             459..536
                     /codon_start=1
                     /label=CBP
                     /note="calmodulin-binding peptide"
                     /translation="KRRWKKNFIAVSAANRFKKISSSGAL"
     CDS             576..596
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="ENLYFQG"
     CDS             647..664
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      731..778
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      815..1270
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        1297..1401
                     /label=AmpR promoter
     CDS             1402..2259
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      2433..3021
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(3207..3349)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(3454..3642)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(4417..4438)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(4454..5533)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(join(5534..5552,1..59))
                     /label=lacI promoter
ORIGIN
        1 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
       61 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
      121 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
      181 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
      241 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
      301 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc
      361 gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa
      421 ataattttgt ttaactttaa gaaggagata tacatatgaa gcgacgatgg aaaaagaatt
      481 tcatagccgt ctcagcagcc aaccgcttta agaaaatctc atcctccggg gcacttagct
      541 cgagcggaac cgcctcgggc ggtgcaacca cgtcagagaa tctctacttc caaggtacct
      601 cggactctag agtcgacctg caggcaagct tgcggccgca ctcgagcacc accaccacca
      661 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
      721 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
      781 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
      841 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
      901 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
      961 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     1021 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     1081 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     1141 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     1201 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     1261 tttacaattt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     1321 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     1381 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     1441 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     1501 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     1561 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     1621 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     1681 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     1741 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     1801 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     1861 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     1921 gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact
     1981 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     2041 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     2101 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     2161 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     2221 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     2281 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     2341 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     2401 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     2461 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     2521 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     2581 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     2641 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     2701 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     2761 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     2821 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     2881 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     2941 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     3001 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     3061 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     3121 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     3181 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     3241 ggtatttcac accgcatata tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     3301 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     3361 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     3421 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     3481 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     3541 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     3601 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     3661 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     3721 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     3781 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     3841 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     3901 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     3961 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     4021 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     4081 cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt
     4141 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     4201 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     4261 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     4321 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     4381 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     4441 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4501 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     4561 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     4621 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     4681 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     4741 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     4801 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     4861 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     4921 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     4981 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     5041 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     5101 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     5161 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     5221 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     5281 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     5341 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     5401 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     5461 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     5521 ttactggttt cacattcacc accctgaatt ga
//
