LOCUS       40924_18346        6042 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pET21-vioE, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6042)
  AUTHORS   Pardee K, Slomovic S, Nguyen PQ, Lee JW, Donghia N, Burrill D, 
            Ferrante T, McSorley F, Furuta Y, Lewandowski M, Boddy CN, Joshi NS,
            Collins JJ.
  TITLE     Portable, On-demand Biomolecular Manufacturing
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6042)
  AUTHORS   Pardee K, Slomovic S, Nguyen PQ, Lee JW, Donghia N, Burrill D, 
            Ferrante T, McSorley F, Furuta Y, Lewandowski M, Boddy CN, Joshi NS,
            Collins JJ.
  TITLE     Direct Submission
  JOURNAL   Submitted (29-JUN-2016) Wyss Institute, Harvard University, 3 
            Blackfan Circle, Wyss Institute Rm517-1D, Boston, MA 02115, USA
REFERENCE   3  (bases 1 to 6042)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6042)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (29-JUN-2016) Wyss Institute, Harvard University, 3 Blackfan Circle,
            Wyss Institute Rm517-1D, Boston, MA 02115, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..6042
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..467
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        493..597
                     /label=AmpR promoter
     CDS             598..1455
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      1629..2217
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(2403..2545)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(2650..2838)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(3613..3634)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        complement(4729..4806)
                     /label=lacI promoter
     promoter        5115..5133
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5134..5158
                     /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             5173..5195
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5265..5285
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="ENLYFQG"
     CDS             5295..5318
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     terminator      5970..6017
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
      481 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
      541 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
      601 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
      661 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
      721 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
      781 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
      841 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
      901 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
      961 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     1021 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     1081 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     1141 gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     1201 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     1261 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     1321 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     1381 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     1441 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     1501 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     1561 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     1621 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     1681 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     1741 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     1801 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     1861 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     1921 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     1981 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     2041 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     2101 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     2161 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     2221 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     2281 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     2341 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     2401 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatatggt
     2461 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     2521 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     2581 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     2641 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     2701 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     2761 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     2821 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     2881 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     2941 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     3001 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     3061 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     3121 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     3181 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     3241 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     3301 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     3361 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     3421 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     3481 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     3541 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     3601 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     3661 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     3721 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     3781 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     3841 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     3901 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
     3961 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     4021 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     4081 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     4141 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     4201 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     4261 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     4321 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     4381 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     4441 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     4501 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     4561 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     4621 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa cggtctgata
     4681 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     4741 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     4801 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     4861 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     4921 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     4981 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     5041 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     5101 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     5161 tctagtaata attttgttta actttaagaa ggagatccac catgggtcta gactcttcta
     5221 gcggcggagg cggaagcgga ggatctggcg gagggggatc aggcgagaac ctgtattttc
     5281 aaggcatggc cagcgactac aaggacgacg acgacaaaca tatggaaaac cgggaaccgc
     5341 cgctgctgcc ggcgcgctgg agcagcgcct atgtgtcgta ctggagtccg atgctgccgg
     5401 atgaccagct gacgtccggc tactgctggt tcgactacga gcgcgacatc tgtcggatag
     5461 acggcctgtt caatccctgg tcggagcgcg acaccggcta ccggctgtgg atgtccgagg
     5521 tcggcaacgc cgccagcggc cgcacctgga agcagaaggt ggcctatggc cgcgagcgga
     5581 ccgccctggg cgagcagctg tgcgagcggc cgctggacga cgagaccggc ccgttcgccg
     5641 agctgttcct gccgcgcgac gtgctgcgcc ggctgggcgc ccgccatatc ggccgccgcg
     5701 tggtgctggg cagggaagcc gacggctggc gctaccagcg tccgggcaag gggccgtcca
     5761 cgttgtacct ggacgccgcc agcggtacgc cgctgaggat ggtgaccggg gacgaggcgt
     5821 cgcgcgcgtc gctgcgcgat ttccccaacg tcagcgaggc cgagattccc gtcttcgccg
     5881 ccaagcgcga attcctaggc tgataagatc cggctgctaa caaagcccga aaggaagctg
     5941 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     6001 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg at
//
