LOCUS       V016307                 8785 bp    DNA     circular SYN 16-MAY-2020
DEFINITION  Exported.
ACCESSION   V016307
VERSION     V016307
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 8785)
  AUTHORS   Wang Q.
  TITLE     Direct Submission
  JOURNAL   Submitted (20-FEB-2020) Single-Cell Center, Qingdao Institute of
            Bioenergy and Bioprocess Technology, No. 189 Songling Road, Laoshan
            District, Qingdao, Shandong Province, Qindao, Shandong 266000, China
REFERENCE   2  (bases 1 to 8785)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing
            ##Assembly-Data-END##
            SGRef: number: 1; type: "Journal Article"; journalName: "Submitted
            (20-FEB-2020) Single-Cell Center, Qingdao Institute of Bioenergy and
            Bioprocess Technology, No. 189 Songling Road, Laoshan District,
            Qingdao, Shandong Province, Qindao, Shandong 266000, China"
FEATURES             Location/Qualifiers
     source          1..8785
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA
                     polymerase"
     CDS             1564..2559
                     /gene="hyg"
                     /label="Hygromycin-B 7''-O-kinase"
                     /note="Hygromycin-B 7''-O-kinase from Streptomyces
                     hygroscopicus. Accession#: P09979"
     CDS             2560..2577
                     /label="6xHis"
                     /note="6xHis affinity tag"
     misc_RNA        3260..3335
                     /label="gRNA scaffold"
                     /note="guide RNA scaffold for the Streptococcus pyogenes
                     CRISPR/Cas9 system"
     CDS             complement(3623..3655)
                     /label="T7 tag (gene 10 leader)"
                     /note="leader peptide from bacteriophage T7 gene 10"
     CDS             complement(3659..3676)
                     /label="thrombin site"
                     /note="thrombin recognition and cleavage site"
     CDS             complement(3686..3703)
                     /label="6xHis"
                     /note="6xHis affinity tag"
     RBS             complement(3722..3744)
                     /label="RBS"
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(3759..3783)
                     /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(3784..3802)
                     /label="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        4111..4188
                     /label="lacI promoter"
     CDS             4189..5268
                     /label="lacI"
                     /note="lac repressor"
     protein_bind    5284..5305
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             6080..6268
                     /label="rop"
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    6373..6515
                     /label="bom"
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(6701..7289)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             7411..8223
                     /label="KanR"
                     /note="aminoglycoside phosphotransferase"
     rep_origin      complement(8319..8774)
                     /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 ggaagcttgc atcctgcgtc ggtttggcgg
      181 tcttttgtcc tttcctctat agcccgcccg tctagagggc acacgcgatg atctttatat
      241 ctcttcatgt gtctttgttt taactaggat actgccgggt gaatgcccat cagacaagag
      301 gccaaactct atctacaccc ttttgacttc tgttgtggtc gtagtgtgtg cttgcatgcc
      361 ctgaaagtcc aggcatccca cttgtgctct aaccccattc aaaacagcag aagtgcttaa
      421 ttaagatata gattcatgat ctcctgtccc ctccttctta ccttttcaca aacctcacac
      481 agaagtctcc actcttcgcc tctaaaacct ctttttaaat tggtaccgga tcccaggagg
      541 agggagtgaa gaggagaagg gatctggttt cagagatccc cacttctgcc gtcgtctttc
      601 ggccttcctt ccttttaggt gtcatgcctt aggtccttca agtcctcacc tgtcgtcgtc
      661 atgtgtgtgt gtgcccgtca tacaagtcac tcgatccaat tcacgcatcg gttcaatcaa
      721 aataagacta gaccccgagg gaagaagggc agaaggaaat cgaaggggtg ggatgtgtgt
      781 gagagaggga aggagaaatg aaagaagtga acaatgtcat ggtagccagt aaggagagag
      841 tagaagcgaa gaaagcaaaa gcactgttgt gaagaaacga aatggaagat ggtcatcgct
      901 cctggctcta cttgtggttt ttctatcttt aatttcaggc gtcctggttt cgttacatca
      961 gctcccttat ctcattggtt tatcccctac tctactgctg cttcttcctt ccatccgtga
     1021 ctgtataaca acgaattgta gtaccgcaga tagacaatag aaaaatgcca aaaaaggcat
     1081 cattgatttg ctcctcccca ttaagtcact gtacgccacc atcgccacta ccctgagctg
     1141 agctcctact ccttttatac gccttgacac acaaagcacc tagtatgttg tgcgggaccg
     1201 gcgtatgcgt gggcagagag gcgcggtcat ggcgtttgtg tgagcgagcc gcgccctcca
     1261 gctttgtgaa tttttcgtga aggcgaaaac gtgcctatca cactatccca cacgcctaca
     1321 aacaagccac atagcaacaa cgcgagtaca cttatgtttc ctacgttcgt gcccgcacga
     1381 aggggcgtct gtgaaggcgc agggctactc tgggccctcc aaatgatgtg cacgcttccc
     1441 gtctcacttt acacacttcc tcttcactcc ttccgttcac atcaacacac gcacaactta
     1501 agcacacatc agtcagcacc ccttcaacac actcctccct ccaactagtc tcaacccccg
     1561 cggatgaccc aggagtcttt gttgctcctc gatcgcatcg attccgacga ctcctacgcc
     1621 tctctccgca acgaccagga gttctgggag cctctcgctc gccgcgccct cgaggagctc
     1681 ggcctccccg tgccccctgt cctccgcgtc cccggtgagt ccactaaccc cgtgctcgtc
     1741 ggggagcccg accctgtcat caagctcttc ggtgagcact ggtgcggtcc tgagtccctc
     1801 gcttccgaga gcgaggcgta cgctgtcctc gcggacgccc ccgtgcctgt tccccgcttg
     1861 ctcggtcgcg gcgagttgcg ccccggcact ggcgcctggc cctggcccta cttggtcatg
     1921 tcccgcatga ccggcaccac ttggcgctcc gcaatggacg gcaccaccga ccgcaacgcc
     1981 ttgctcgccc tcgcgcgtga gttgggccgc gttctcggcc gtttgcaccg cgtccccctc
     2041 accggcaaca cggtcctcac ccctcactcc gaggtgttcc ccgagctctt gcgcgagcgc
     2101 cgtgccgcga ctgtcgagga ccaccgtggc tggggttacc tctcccctcg cctcctcgac
     2161 cgcttggagg actggctccc cgatgtggac accttgctcg ccggccgcga gcctcgcttc
     2221 gtccacggcg atctccacgg caccaacatt ttcgtcgacc tcgccgctac ggaggtcacg
     2281 ggcatcgtgg atttcaccga cgtctacgcc ggcgactccc gctactccct cgtgcagctc
     2341 cacctcaacg catttcgcgg cgaccgcgag atcttggcgg ccctcttgga cggggcccag
     2401 tggaagcgta ccgaggactt cgcgcgcgag ctcttggctt tcacgttttt gcacgacttc
     2461 gaggttttcg aggagacgcc cttggacttg agcggcttca cggaccccga ggagctcgca
     2521 cagttcctct ggggtcctcc cgacaccgcc cccggggcgc accaccacca ccaccactaa
     2581 ccgcgggttt gtttgtgctg attgcttggt atgagagtgt tgaattcctg catcatgttt
     2641 ttctctgtag tcctttccta cccccgtcat tttcttttct ccctggttct tcttttgtca
     2701 cccttatttt acataaaatt ttctttgttt atagtgagag gaaggtagag aggggaaaac
     2761 aagaacaacg aacgcaagcg tgtgaaagga gggcgaatag aagagaaaca gatctgttga
     2821 gcattgagag tggagccggg ggaaaggctt gtgtgttgtt tttgaaaaag ttgtttaaat
     2881 cacgaatccg ttagttctca tgtgtacctc tttcactaca tgtgatggag aaaacaaaag
     2941 tgtgaggatt aattgaagaa aaagaagagt tcgacacgtc aaaccgccca aaagacgtca
     3001 caaagagaac ttgattctct ttgccgtgtt gatcttgtct tttcccccag cttttcttgc
     3061 cacccgttct agattaagct ggagtgggaa ggcgtggact gtcgctgggc gaatttagaa
     3121 agcgacagca tcgtcttgca tctccgtcct tcctgtcctt attccgcacg caaagttaaa
     3181 caccataaca tcactcatca tccttcacac acatacgcgc actccaaacg cctcatatcg
     3241 agaccaacac aaggtctcag ttttagagct agaaatagca agttaaaata aggctagtcc
     3301 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttaggaagga agaagaatac
     3361 gaagatgggt gatgatgatg ataatggttg gtagacggtt tctctctctt tccccttctc
     3421 tctttctttt ttttaagtga aaaataaaaa cccggaaacg gagacaacga cagcaatatc
     3481 atggcgacgt cgacgaaggg gtaggaaaaa tggagtggag agaggaagga aaaaggccaa
     3541 cccagggtgc ggtgagaggt agggagatag agaggaaggg gaaagtcacg caagcaatca
     3601 tatcataggt gtctttctgc agacccattt gctgtccacc agtcatgcta gccatatggc
     3661 tgccgcgcgg caccaggccg ctgctgtgat gatgatgatg atggctgctg cccatggtat
     3721 atctccttct taaagttaaa caaaattatt tctagagggg aattgttatc cgctcacaat
     3781 tcccctatag tgagtcgtat taatttcgcg ggatcgagat ctcgatcctc tacgccggac
     3841 gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc tggcgcctat atcgccgaca
     3901 tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg
     3961 gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg catgcaccat
     4021 tccttgcggc ggcggtgctc aacggcctca acctactact gggctgcttc ctaatgcagg
     4081 agtcgcataa gggagagcgt cgagatcccg gacaccatcg aatggcgcaa aacctttcgc
     4141 ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta
     4201 acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg
     4261 aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag
     4321 ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt
     4381 ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa
     4441 tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc
     4501 gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt
     4561 aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg
     4621 gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa
     4681 gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg
     4741 ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat
     4801 ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc
     4861 ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt
     4921 gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt
     4981 ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg
     5041 ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg
     5101 ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg
     5161 aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     5221 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     5281 aattaatgta agttagctca ctcattaggc accgggatct cgaccgatgc ccttgagagc
     5341 cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg ccgcacttat
     5401 gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct gggtcatttt
     5461 cggcgaggac cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg cggtattcgg
     5521 aatcttgcac gccctcgctc aagccttcgt cactggtccc gccaccaaac gtttcggcga
     5581 gaagcaggcc attatcgccg gcatggcggc cccacgggtg cgcatgatcg tgctcctgtc
     5641 gttgaggacc cggctaggct ggcggggttg ccttactggt tagcagaatg aatcaccgat
     5701 acgcgagcga acgtgaagcg actgctgctg caaaacgtct gcgacctgag caacaacatg
     5761 aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag cgccctgcac
     5821 cattatgttc cggatctgca tcgcaggatg ctgctggcta ccctgtggaa cacctacatc
     5881 tgtattaacg aagcgctggc attgaccctg agtgattttt ctctggtccc gccgcatcca
     5941 taccgccagt tgtttaccct cacaacgttc cagtaaccgg gcatgttcat catcagtaac
     6001 ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt acccccatga acagaaatcc
     6061 cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt
     6121 tatcagaagc cagacattaa cgcttctgga gaaactcaac gagctggacg cggatgaaca
     6181 ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca gctgcctcgc
     6241 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     6301 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     6361 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt
     6421 aactatgcgg catcagagca gattgtactg agagtgcacc atatatgcgg tgtgaaatac
     6481 cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg
     6541 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     6601 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     6661 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     6721 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     6781 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     6841 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     6901 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     6961 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     7021 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     7081 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     7141 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     7201 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     7261 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     7321 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgaacaat aaaactgtct
     7381 gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcttgc
     7441 tctaggccgc gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc
     7501 gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca
     7561 gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc
     7621 agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact
     7681 cctgatgatg catggttact caccactgcg atccccggga aaacagcatt ccaggtatta
     7741 gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg
     7801 ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct
     7861 caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt
     7921 aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc attctcaccg
     7981 gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa
     8041 ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc
     8101 atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa
     8161 tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt
     8221 ttctaagaat taattcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     8281 aggggttccg cgcacatttc cccgaaaagt gccacctgaa attgtaaacg ttaatatttt
     8341 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     8401 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     8461 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     8521 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     8581 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     8641 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     8701 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     8761 gctacagggc gcgtcccatt cgcca
//
