LOCUS       40924_18361        8043 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Bacterial expression of tandem high affinity maltose binding protein
            domains linked to SpyCatcher, for solid-phase polyprotein 
            synthesis..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8043)
  AUTHORS   Veggiani G, Nakamura T, Brenner MD, Gayet RV, Yan J, Robinson CV, 
            Howarth M
  TITLE     Programmable polyproteams built using twin peptide superglues.
  JOURNAL   Proc Natl Acad Sci U S A. 2016 Jan 19. pii: 201519214.
  PUBMED    26787909
REFERENCE   2  (bases 1 to 8043)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8043)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A. 2016 Jan 19. pii: 201519214."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8043
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(140..157)
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             complement(2783..2800)
                     /codon_start=1
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
                     /translation="LVPRGS"
     CDS             complement(2810..2827)
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     RBS             complement(2847..2869)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(2884..2908)
                     /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)."
     promoter        complement(2909..2927)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3011..3030)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI,
                     reverse primer"
     promoter        3236..3313
                     /label=lacI promoter
     CDS             3314..4393
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    4409..4430
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             5205..5393
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5390..5412)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5498..5640
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5655..5672)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5826..6414)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6588..7445)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7446..7550)
                     /label=AmpR promoter
     rep_origin      complement(7577..8032)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttct
      181 attaaatatg agcgtcacct ttagttgctt tgccatttac agtaacctga ccttgctcat
      241 taactgtaaa ggtaatagca gttgctacct cataaccgtc tggtgctgcg gtttcgacaa
      301 atgtatattt tcctggatac aggtagaaat ctttcacttg tccatctgaa atccatgtac
      361 taatagtttt accagatgaa tcacgcaact ccatagttgc accagctaac tctttgccgt
      421 cctcatcacg ttttgagaat ttaatatggg tagcactatc ttcttcaatt gtcatatcac
      481 cggactgacc ttgctcactt gataaacctg ataaggtatc aaccatggca ccactaccgc
      541 ccgaacccga gctcgaatta gtctgcgcgt ctttcagggc ttcatcgaca gtctgacgac
      601 cgctggcggc gttgatcacc gcagtacgca cggcatacca gaaagcggac atctgcggga
      661 tgttcggcat gatttcacct ttctgggcgt tttccatagt ggcggccaca cgtggatctt
      721 tcaccaactc ttcctcgtaa gacttcagcg ctacggcacc cagcggtttg tctttattaa
      781 ccgcttccag accttcatca gtcagcagat agttttcgag gaactctttt gccagctctt
      841 tgttcggact ggcggcgtta atacctgcgc tcagcacgcc aacgaacggt ttggatggtt
      901 gacccttgaa ggtcggcagt accgttacac cataattcac tttgctggtg tcgatgttgg
      961 accatgccca cgggccgttg atggtcatcg ctgtttcgcc tttattaaag gcagcttctg
     1021 cgatggagta atcggtgtct gcattcatgt gtttgttttt aatcaggtca accaggaagg
     1081 tcagacccgc tttcgcgcca gcgttatcca cgcccacgtc tttaatgtcg ccatacttga
     1141 acgcataacc cccgtcagca gcaatcagcg gccaggtgaa gtacggttct tgcaggttga
     1201 acatcagcgc gctcttacct ttcgctttca gttctttatc cagcgccggg atctcttccc
     1261 aggtttttgg cgggttcggc agcagatctt tgttataaat cagcgataac gcttcaacag
     1321 cgatcgggta agcaatcagc ttgccgttgt aacgtacggc atcccaggta aacggataca
     1381 gcttgtcctg gaacgctttg tccggggtga tttcagccaa caggccagat tgagcgtagc
     1441 caccaaagcg gtcgtgtgcc cagaagataa tgtcagggcc atcgccagtt gccgcaacct
     1501 gtgggaattt ctcttccagt ttatccggat gctcaacggt gactttaatt ccggtatctt
     1561 tctcgaattt cttaccgact tcagcgagac cgttatagcc tttatcgccg ttaatccaga
     1621 ttaccagttt accctcctct atctttccgg atccacctcc ggatccgccg gaactagaat
     1681 tcgtctgcgc gtctttcagg gcttcatcga cagtctgacg accgctggcg gcgttgatca
     1741 ccgcagtacg cacggcatac cagaaagcgg acatctgcgg gatgttcggc atgatttcac
     1801 ctttctgggc gttttccata gtggcggcca cacgtggatc tttcaccaac tcttcctcgt
     1861 aagacttcag cgctacggca cccagcggtt tgtctttatt aaccgcttcc agaccttcat
     1921 cagtcagcag atagttttcg aggaactctt ttgccagctc tttgttcgga ctggcggcgt
     1981 taatacctgc gctcagcacg ccaacgaacg gtttggatgg ttgacccttg aaggtcggca
     2041 gtaccgttac accataattc actttgctgg tgtcgatgtt ggaccatgcc cacgggccgt
     2101 tgatggtcat cgctgtttcg cctttattaa aggcagcttc tgcgatggag taatcggtgt
     2161 ctgcattcat gtgtttgttt ttaatcaggt caaccaggaa ggtcagaccc gctttcgcgc
     2221 cagcgttatc cacgcccacg tctttaatgt cgccatactt gaacgcataa cccccgtcag
     2281 cagcaatcag cggccaggtg aagtacggtt cttgcaggtt gaacatcagc gcgctcttac
     2341 ctttcgcttt cagttcttta tccagcgccg ggatctcttc ccaggttttt ggcgggttcg
     2401 gcagcagatc tttgttataa atcagcgata acgcttcaac agcgatcggg taagcaatca
     2461 gcttgccgtt gtaacgtacg gcatcccagg taaacggata cagcttgtcc tggaacgctt
     2521 tgtccggggt gatttcagcc aacaggccag attgagcgta gccaccaaag cggtcgtgtg
     2581 cccagaagat aatgtcaggg ccatcgccag ttgccgcaac ctgtgggaat ttctcttcca
     2641 gtttatccgg atgctcaacg gtgactttaa ttccggtatc tttctcgaat ttcttaccga
     2701 cttcagcgag accgttatag cctttatcgc cgttaatcca gattaccagt ttaccttctt
     2761 cgattttcat gctagccata tggctgccgc gcggcaccag gccgctgctg tgatgatgat
     2821 gatgatggct gctgcccata tgtatatctc cttcttaaag ttaaacaaaa ttatttctag
     2881 aggggaattg ttatccgctc acaattcccc tatagtgagt cgtattaatt tcgcgggatc
     2941 gagatctcga tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg
     3001 gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg
     3061 ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg
     3121 ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta
     3181 ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgaga tcccggacac
     3241 catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt
     3301 cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc
     3361 ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga
     3421 aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact
     3481 ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc
     3541 gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt
     3601 gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc
     3661 gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt
     3721 ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat
     3781 caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc
     3841 attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct
     3901 gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg
     3961 ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg
     4021 catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc
     4081 cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac
     4141 cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct
     4201 ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa
     4261 tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac
     4321 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     4381 ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcaccgg
     4441 gatctcgacc gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg
     4501 gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg
     4561 tgccggcagc gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga
     4621 tcggcctgtc gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg
     4681 gtcccgccac caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccccac
     4741 gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg ggttgcctta
     4801 ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa
     4861 cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg
     4921 aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca ggatgctgct
     4981 ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga ccctgagtga
     5041 tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa cgttccagta
     5101 accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt ttcatcggta
     5161 tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc aaacaggaaa
     5221 aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt ctggagaaac
     5281 tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac gaccacgctg
     5341 atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     5401 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     5461 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta
     5521 gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt
     5581 gcaccatata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     5641 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     5701 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5761 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5821 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5881 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5941 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     6001 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     6061 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     6121 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     6181 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     6241 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     6301 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     6361 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     6421 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6481 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     6541 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     6601 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     6661 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     6721 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6781 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6841 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6901 gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6961 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     7021 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     7081 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     7141 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     7201 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     7261 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     7321 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     7381 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     7441 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     7501 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     7561 cgaaaagtgc cacctgaaat tgtaaacgtt aatattttgt taaaattcgc gttaaatttt
     7621 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     7681 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     7741 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     7801 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     7861 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     7921 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     7981 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg
     8041 cca
//
