LOCUS       62056_10155        5377 bp DNA     circular SYN 02-OCT-2019
DEFINITION  Cloning vector pET28AMP_MaIE, complete sequence.
ACCESSION   MK606522
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5377)
  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 5377)
  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..5377
                     /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"
     RBS             complement(314..336)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(351..375)
                     /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(376..394)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        703..780
                     /label=lacI promoter
     CDS             781..1860
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    1876..1897
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             2672..2860
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    2965..3107
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3293..3881)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             4003..4815
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      complement(4911..5366)
                     /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 gtgaatttta gccagtgcgg
      241 aagcagagaa catcatggta gtcagtgcgg acagtgccag gatacgagca ccggttttga
      301 ttttcatggt atatctcctt cttaaagtta aacaaaatta tttctagagg ggaattgtta
      361 tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag atctcgatcc
      421 tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct
      481 atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt
      541 gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct
      601 tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct
      661 tcctaatgca ggagtcgcat aagggagagc gtcgagatcc cggacaccat cgaatggcgc
      721 aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat
      781 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt
      841 tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg
      901 gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag
      961 tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc
     1021 gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa
     1081 cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt
     1141 gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc
     1201 actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt
     1261 ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag
     1321 caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc
     1381 tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg
     1441 agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact
     1501 gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc
     1561 gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca
     1621 tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc
     1681 gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc
     1741 gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc
     1801 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     1861 tgagcgcaac gcaattaatg taagttagct cactcattag gcaccgggat ctcgaccgat
     1921 gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt
     1981 cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct
     2041 ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct
     2101 tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa
     2161 acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccccacggg tgcgcatgat
     2221 cgtgctcctg tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa
     2281 tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg
     2341 agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc
     2401 agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg
     2461 aacacctaca tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc
     2521 ccgccgcatc cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc
     2581 atcatcagta acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat
     2641 gaacagaaat cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa
     2701 catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga
     2761 cgcggatgaa caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg
     2821 cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     2881 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     2941 agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt
     3001 gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatatgc
     3061 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcctccgctt
     3121 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     3181 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     3241 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     3301 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3361 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     3421 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     3481 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     3541 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     3601 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     3661 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     3721 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     3781 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     3841 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     3901 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgaaca
     3961 ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcaacgg
     4021 gaaacgtctt gctctaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat
     4081 aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag
     4141 cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca
     4201 gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat
     4261 tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg gaaaacagca
     4321 ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg
     4381 ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta
     4441 tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt
     4501 gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg
     4561 ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt
     4621 gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac
     4681 caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg
     4741 ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg
     4801 ctcgatgagt ttttctaaga attaattcat gagcggatac atatttgaat gtatttagaa
     4861 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg aaattgtaaa
     4921 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     4981 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     5041 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     5101 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     5161 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     5221 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     5281 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     5341 gcttaatgcg ccgctacagg gcgcgtccca ttcgcca
//
