LOCUS       62056_17800        5979 bp DNA     circular SYN 10-JUN-2019
DEFINITION  Expression vector pNC-ET28, complete sequence.
ACCESSION   MK720606
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5979)
  AUTHORS   Yan P, Zeng Y, Shen W, Tuo D, Zhou P.
  TITLE     Direct Submission
  JOURNAL   Submitted (31-MAR-2019) Institute of Tropical Bioscience and 
            Biotechnology, Chinese Academy of Tropical Agriculture Sciences, 
            Xueyuan Road, Haikou, Hainan 571101, China
REFERENCE   2  (bases 1 to 5979)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Submitted 
            (31-MAR-2019) Institute of Tropical Bioscience and Biotechnology, 
            Chinese Academy of Tropical Agriculture Sciences, Xueyuan Road, 
            Haikou, Hainan 571101, China"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..5979
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        182..284
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     CDS             315..617
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     CDS             696..713
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      780..827
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      864..1319
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(1415..2227)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      2349..2937
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(3123..3265)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(3370..3558)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(4333..4354)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(4370..5449)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(5450..5527)
                     /label=lacI promoter
     promoter        5836..5854
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5855..5879
                     /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             5894..5916
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5935..5952
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             5962..5979
                     /codon_start=1
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
                     /translation="LVPRGS"
ORIGIN
        1 cagtggtctc tgtccagtcc tggcctcgtc ggccattctc gactaagttg gcagcatcac
       61 ccgacgcact ttgcgccgaa taaatacctg tgacggaaga tcacttcgca gaataaataa
      121 atcctggtgt ccctgttgat accgggaagc cctgggccaa cttttggcga aaatgagacg
      181 ttgatcggca cgtaagaggt tccaactttc accataatga aataagatca ctaccgggcg
      241 tattttttga gttatcgaga ttttcaggag ctaaggaagc taaacttttg ctgacgagaa
      301 cagggactgg tgaaatgcag tttaaggttt acacctataa aagagagagc cgttatcgtc
      361 tgtttgtgga tgtacagagt gatattattg acacgcctgg gcgacggatg gtgatccccc
      421 tggccagtgc acgtctgctg tcagataaag tctcccgtga actttacccg gtggtgcata
      481 tcggggatga aagctggcgc atgatgacca ccgatatggc cagtgtgccg gtatccgtta
      541 tcggggaaga agtggctgat ctcagccacc gcgaaaatga catcaaaaac gccattaacc
      601 tgatgttctg gggaatataa atgtcaggct cccttataca cagggccagt ctggccactt
      661 gtggtctgct gagaccgctt gcggccgcac tcgagcacca ccaccaccac cactgagatc
      721 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
      781 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
      841 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
      901 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
      961 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     1021 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     1081 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     1141 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     1201 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     1261 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc
     1321 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     1381 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     1441 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     1501 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     1561 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     1621 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     1681 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     1741 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     1801 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     1861 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     1921 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     1981 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     2041 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     2101 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     2161 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     2221 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     2281 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     2341 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     2401 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     2461 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     2521 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     2581 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     2641 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     2701 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     2761 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     2821 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     2881 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     2941 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     3001 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     3061 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     3121 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatatggt
     3181 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     3241 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     3301 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     3361 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     3421 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     3481 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     3541 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     3601 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     3661 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     3721 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     3781 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     3841 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     3901 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     3961 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     4021 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     4081 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     4141 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     4201 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     4261 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     4321 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     4381 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4441 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     4501 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     4561 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     4621 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
     4681 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     4741 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     4801 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     4861 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     4921 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     4981 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     5041 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     5101 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     5161 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     5221 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     5281 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     5341 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     5401 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     5461 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     5521 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     5581 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     5641 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     5701 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     5761 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     5821 tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc
     5881 ctctagaaat aattttgttt aactttaaga aggagatata ccatgggcag cagccatcat
     5941 catcatcatc acagcagcgg cctggtgccg cgcggcagc
//
