LOCUS       62056_10180        5359 bp DNA     circular SYN 02-OCT-2019
DEFINITION  Cloning vector pET28AMP_PhoA, complete sequence.
ACCESSION   MK606526
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5359)
  AUTHORS   Skowron PM, Zylicz-Stachula A.
  TITLE     Direct Submission
  JOURNAL   Submitted (07-MAR-2019) University of Gdansk, Faculty of Chemistry, 
            Wita Stwosza 63, Gdansk, Pomorskie 80-308, Poland
REFERENCE   2  (bases 1 to 5359)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Submitted 
            (07-MAR-2019) University of Gdansk, Faculty of Chemistry, Wita 
            Stwosza 63, Gdansk, Pomorskie 80-308, Poland"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..5359
                     /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(206..223)
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     sig_peptide     complement(227..289)
                     /label=phoA signal sequence
                     /note="signal sequence of E. coli alkaline phosphatase
                     (Inouye et al., 1982)"
     RBS             complement(296..318)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(333..357)
                     /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(358..376)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        685..762
                     /label=lacI promoter
     CDS             763..1842
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    1858..1879
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             2654..2842
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    2947..3089
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3275..3863)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             3985..4797
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      complement(4893..5348)
                     /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 gagcttactt acttaccatg
      181 ctcttctggg cccgggtgaa gagctgtggt ggtgatggtg gtggcgtgct ttggtaaccg
      241 gagtgaacag cagcggcagc agagccagtg cgatggtgga ctgtttcatg gtatatctcc
      301 ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca caattcccct
      361 atagtgagtc gtattaattt cgcgggatcg agatctcgat cctctacgcc ggacgcatcg
      421 tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg
      481 atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg
      541 tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca ccattccttg
      601 cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg caggagtcgc
      661 ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt tcgcggtatg
      721 gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta
      781 tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag
      841 gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat
      901 tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt
      961 gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc
     1021 gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc
     1081 tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat
     1141 ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta
     1201 tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt
     1261 acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg
     1321 ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact
     1381 cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt
     1441 caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac
     1501 gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg
     1561 gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtta
     1621 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa
     1681 ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga
     1741 aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     1801 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     1861 tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga gagccttcaa
     1921 cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac ttatgactgt
     1981 cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca ttttcggcga
     2041 ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat tcggaatctt
     2101 gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca
     2161 ggccattatc gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc tgtcgttgag
     2221 gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga
     2281 gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt
     2341 cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat
     2401 gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt
     2461 aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc
     2521 cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat
     2581 cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta
     2641 cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag
     2701 aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga
     2761 catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc tcgcgcgttt
     2821 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     2881 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     2941 tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat
     3001 gcggcatcag agcagattgt actgagagtg caccatatat gcggtgtgaa ataccgcaca
     3061 gatgcgtaag gagaaaatac cgcatcaggc gctcctccgc ttcctcgctc actgactcgc
     3121 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     3181 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     3241 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3301 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3361 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3421 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3481 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3541 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     3601 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     3661 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     3721 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     3781 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     3841 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     3901 agtggaacga aaactcacgt taagggattt tggtcatgaa caataaaact gtctgcttac
     3961 ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc ttgctctagg
     4021 ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat
     4081 gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg
     4141 tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta
     4201 aactggctga cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat
     4261 gatgcatggt tactcaccac tgcgatcccc gggaaaacag cattccaggt attagaagaa
     4321 tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat
     4381 tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg
     4441 caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc
     4501 tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca
     4561 gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata
     4621 ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta
     4681 tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt
     4741 attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa
     4801 gaattaattc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     4861 tccgcgcaca tttccccgaa aagtgccacc tgaaattgta aacgttaata ttttgttaaa
     4921 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     4981 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     5041 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     5101 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     5161 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     5221 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     5281 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     5341 gggcgcgtcc cattcgcca
//
