LOCUS       62056_10175        7543 bp DNA     circular SYN 02-OCT-2019
DEFINITION  Cloning vector pET28AMP_MaIE_AGF40, complete sequence.
ACCESSION   MK606525
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7543)
  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 7543)
  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..7543
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(13..30)
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     RBS             complement(121..143)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(158..182)
                     /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(183..201)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        510..587
                     /label=lacI promoter
     CDS             588..1667
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    1683..1704
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             2479..2667
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    2772..2914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3100..3688)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             3810..4622
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      complement(4718..5173)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     terminator      complement(5210..5257)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(5324..5341)
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
ORIGIN
        1 gggtgaagag ctgtggtggt gatggtggtg aattttagcc agtgcggaag cagagaacat
       61 catggtagtc agtgcggaca gtgccaggat acgagcaccg gttttgattt tcatggtata
      121 tctccttctt aaagttaaac aaaattattt ctagagggga attgttatcc gctcacaatt
      181 cccctatagt gagtcgtatt aatttcgcgg gatcgagatc tcgatcctct acgccggacg
      241 catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat
      301 caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg
      361 tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc atgcaccatt
      421 ccttgcggcg gcggtgctca acggcctcaa cctactactg ggctgcttcc taatgcagga
      481 gtcgcataag ggagagcgtc gagatcccgg acaccatcga atggcgcaaa acctttcgcg
      541 gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa
      601 cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga
      661 accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc
      721 tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg
      781 gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat
      841 ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg
      901 aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta
      961 actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg
     1021 cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag
     1081 acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt
     1141 tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc
     1201 tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg
     1261 gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg
     1321 ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg
     1381 gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc
     1441 cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc
     1501 tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga
     1561 aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     1621 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     1681 attaatgtaa gttagctcac tcattaggca ccgggatctc gaccgatgcc cttgagagcc
     1741 ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg
     1801 actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctg ggtcattttc
     1861 ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga
     1921 atcttgcacg ccctcgctca agccttcgtc actggtcccg ccaccaaacg tttcggcgag
     1981 aagcaggcca ttatcgccgg catggcggcc ccacgggtgc gcatgatcgt gctcctgtcg
     2041 ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga atcaccgata
     2101 cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc aacaacatga
     2161 atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc
     2221 attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac acctacatct
     2281 gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg ccgcatccat
     2341 accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc
     2401 cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc
     2461 ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt
     2521 atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag
     2581 gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg
     2641 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     2701 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     2761 gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta
     2821 actatgcggc atcagagcag attgtactga gagtgcacca tatatgcggt gtgaaatacc
     2881 gcacagatgc gtaaggagaa aataccgcat caggcgctcc tccgcttcct cgctcactga
     2941 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     3001 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     3061 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3121 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3181 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3241 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3301 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3361 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3421 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     3481 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     3541 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     3601 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     3661 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     3721 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgaacaata aaactgtctg
     3781 cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcttgct
     3841 ctaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg
     3901 ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag
     3961 agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca
     4021 gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc
     4081 ctgatgatgc atggttactc accactgcga tccccgggaa aacagcattc caggtattag
     4141 aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt
     4201 tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc
     4261 aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta
     4321 atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg
     4381 attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat
     4441 taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca
     4501 tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat
     4561 atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt
     4621 tctaagaatt aattcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     4681 ggggttccgc gcacatttcc ccgaaaagtg ccacctgaaa ttgtaaacgt taatattttg
     4741 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     4801 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     4861 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     4921 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     4981 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     5041 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     5101 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     5161 ctacagggcg cgtcccattc gccaatccgg atatagttcc tcctttcagc aaaaaacccc
     5221 tcaagacccg tttagaggcc ccaaggggtt atgctagtta ttgctcagcg gtggcagcag
     5281 ccaactcagc ttcctttcgg gctttgttag cagccggatc tcagtggtgg tggtggtggt
     5341 gctcgagctt acttacttac catgctcttc tggggccacc aactggtgcc gcgccaccgc
     5401 ccagcaattc gtgatcacca cggctggtac cgcccaccgg agccgcaccg ccacccagga
     5461 gctcgtggtc accacggctg gtgccaccaa ctggggccgc gccaccgccc agaagctcgt
     5521 gatcaccacg gctggtaccg cccactggag ccgcaccgcc acccagaagc tcgtggtcac
     5581 cacggctggt gccaccaacg ggtgccgcgc caccgcccag taattcgtga tcgccacggc
     5641 tggtaccgcc cacgggtgcc gcaccgccac ccagtaactc gtggtcacca cggctggtgc
     5701 caccaacagg agccgcgcca ccgcccagta actcgtgatc gccacggctg gtaccgccca
     5761 caggagccgc accgccaccc agtaattcgt ggtcaccacg gctggtgcca ccaaccggcg
     5821 ccgcgccacc gcccagcagt tcgtgatcgc cacggctggt accgcccacc ggcgccgcac
     5881 cgccacccag cagctcgtgg tcaccacggc tggtggggcc accaactggt gccgcgccac
     5941 cgcccagcaa ttcgtgatca ccacggctgg taccgcccac cggagccgca ccgccaccca
     6001 ggagctcgtg gtcaccacgg ctggtgccac caactggggc cgcgccaccg cccagaagct
     6061 cgtgatcacc acggctggta ccgcccactg gagccgcacc gccacccaga agctcgtggt
     6121 caccacggct ggtgccacca acgggtgccg cgccaccgcc cagtaattcg tgatcgccac
     6181 ggctggtacc gcccacgggt gccgcaccgc cacccagtaa ctcgtggtca ccacggctgg
     6241 tgccaccaac aggagccgcg ccaccgccca gtaactcgtg atcgccacgg ctggtaccgc
     6301 ccacaggagc cgcaccgcca cccagtaatt cgtggtcacc acggctggtg ccaccaaccg
     6361 gcgccgcgcc accgcccagc agttcgtgat cgccacggct ggtaccgccc accggcgccg
     6421 caccgccacc cagcagctcg tggtcaccac ggctggtggg gccaccaact ggtgccgcgc
     6481 caccgcccag caattcgtga tcaccacggc tggtaccgcc caccggagcc gcaccgccac
     6541 ccaggagctc gtggtcacca cggctggtgc caccaactgg ggccgcgcca ccgcccagaa
     6601 gctcgtgatc accacggctg gtaccgccca ctggagccgc accgccaccc agaagctcgt
     6661 ggtcaccacg gctggtgcca ccaacgggtg ccgcgccacc gcccagtaat tcgtgatcgc
     6721 cacggctggt accgcccacg ggtgccgcac cgccacccag taactcgtgg tcaccacggc
     6781 tggtgccacc aacaggagcc gcgccaccgc ccagtaactc gtgatcgcca cggctggtac
     6841 cgcccacagg agccgcaccg ccacccagta attcgtggtc accacggctg gtgccaccaa
     6901 ccggcgccgc gccaccgccc agcagttcgt gatcgccacg gctggtaccg cccaccggcg
     6961 ccgcaccgcc acccagcagc tcgtggtcac cacggctggt ggggccacca actggtgccg
     7021 cgccaccgcc cagcaattcg tgatcaccac ggctggtacc gcccaccgga gccgcaccgc
     7081 cacccaggag ctcgtggtca ccacggctgg tgccaccaac tggggccgcg ccaccgccca
     7141 gaagctcgtg atcaccacgg ctggtaccgc ccactggagc cgcaccgcca cccagaagct
     7201 cgtggtcacc acggctggtg ccaccaacgg gtgccgcgcc accgcccagt aattcgtgat
     7261 cgccacggct ggtaccgccc acgggtgccg caccgccacc cagtaactcg tggtcaccac
     7321 ggctggtgcc accaacagga gccgcgccac cgcccagtaa ctcgtgatcg ccacggctgg
     7381 taccgcccac aggagccgca ccgccaccca gtaattcgtg gtcaccacgg ctggtgccac
     7441 caaccggcgc cgcgccaccg cccagcagtt cgtgatcgcc acggctggta ccgcccaccg
     7501 gcgccgcacc gccacccagc agctcgtggt caccacggct ggt
//
