LOCUS       40924_33322        6737 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pNK076, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6737)
  AUTHORS   Kandul NP, Guo M, Hay BA.
  TITLE     A positive readout single transcript reporter for site-specific mRNA
            cleavage
  JOURNAL   PeerJ (2017) In press
REFERENCE   2  (bases 1 to 6737)
  AUTHORS   Kandul NP, Guo M, Hay BA.
  TITLE     Direct Submission
  JOURNAL   Submitted (02-JAN-2017) Biology and Biological Engineering, 
            California Institute of Technology, 1200 E. California Blvd., 
            Pasadena, CA 91125, USA
REFERENCE   3  (bases 1 to 6737)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6737)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PeerJ 
            (2017) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (02-JAN-2017) Biology and Biological Engineering, California 
            Institute of Technology, 1200 E. California Blvd., Pasadena, CA 
            91125, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..6737
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      4..460
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     601..617
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        627..645
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        699..3208
                     /label=Ac5 promoter
                     /note="Drosophila melanogaster actin 5C promoter"
     CDS             3236..4168
                     /codon_start=1
                     /label=Rluc
                     /note="luciferase from the anthozoan coelenterate Renilla 
                     reniformis (sea pansy)"
                     /translation="MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     promoter        complement(4549..4567)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4588..4604)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4612..4628)
                     /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(4636..4666)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(4681..4702)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(4990..5578)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5752..6609)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6610..6714)
                     /label=AmpR promoter
ORIGIN
        1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
       61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
      121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
      181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
      241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
      301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
      361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa atgagctgat ttaacaaaaa
      421 tttaacgcga attttaacaa aatattaacg cttacaattt ccattcgcca ttcaggctgc
      481 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
      541 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
      601 gtaaaacgac ggccagtgag cgcgcgtaat acgactcact atagggcgaa ttggccacgc
      661 tgaggaaggg ccggccttct taattaacta aaaaaaatca tgaatggcat caactctgaa
      721 tcaaatcttt gcagatgcac ctacttctca tttccactgt cacatcattt ttccagatct
      781 cgctgcctgt tatgtggccc acaaaccaag acacgtttta tggccattaa agctggctga
      841 tcgtcgccaa acaccaaata cataatgaat atgtacacat tcgagaaaga agcgatcaaa
      901 gaagcgtctt cgggcggagt aggagaatgc ggaggagaag gagaacgagc tgatctagta
      961 tctctccaca atccaatgcc aactgaccaa ctggccatat tcggagcaat ttgaagccaa
     1021 tttccatcgc ctggcgatcg ctccattctt ggctatatgt ttttcaccgt tacccggggc
     1081 cattttcaaa gactcgtcgg caagataaga ttgtgtcact cgctgtctct cttcatttgt
     1141 cgaagaatgc tgaggaattt cgcgatgacg tcggcgagta ttttgaagaa tgagaataat
     1201 ttgtatttat acgaaaatca gttagtggaa ttttctacaa aaacatgtta tctatagata
     1261 attttgttgc aaaatatgtt gactatgaca aagattgtat gtatatacct ttaatgtatt
     1321 ctcattttct tatgtattta taatggcaat gatgatactg atgatatttt aagatgatgc
     1381 cagaccaaaa ggcttgaatt tctgcgtctt ttgccgaacg cagtgcatgt gcaattgttg
     1441 ttttttggaa tattcaattt tcggactgtc cgctttgatt tcagtttctt ggcttattca
     1501 aaaagcaaag taaagccaaa aaagcgagat ggcaatacca aatgcggcaa aacggtagtg
     1561 gaaggaaagg ggtgcggggc agcggaagga agggtggggc ggggcgtggc ggggtctgtg
     1621 gctgggcgcg acgtcaccga cgttggagcc actcctttga ccatgtgtgc gtgtgtgtat
     1681 tattcgtgtc tcgccactcg ccggttgttt ttttcttttt atgctgcgct ctctctagcg
     1741 ccatctcgct tacgcatgct caacgcaccg catgttgccg tttcctttta tgcgtcattt
     1801 tggctcgaaa taggcaatta tttaaacaaa gattagtcaa cgaaaacgct aaaataaata
     1861 agtctacaat atggttactt attgccatgt gtgtgcagcc aacgatagca acaaaagcaa
     1921 caacacaggt ggctttccct ctttcacttt ttgtttgcaa gccgcgtgcg agcaagacgg
     1981 cacgaccggc aaacgcaatt acgctgacaa agagcagacg aagttttggc gaaaaacatc
     2041 aaggcgcctg atacgaatgc atttgcaata acaattgcga tatttaatat tgtttatgaa
     2101 gctgtttgac ttcaaaacac acaaaaaaaa aaataaaaca aattatttga aagagaatta
     2161 ggaatcggac gcttatcgtt agggtaacaa caagaaatgc ttactgagtc acagcctctg
     2221 gaaaactgcc gcaagccaga gagagagaga aaaagaggga gagcagctta gaccgcatgt
     2281 gcttgtgtgt gaggcgtctc tctcttcgtc tctgttgcgc aaacgcatag actgcactga
     2341 aaaaatcgat tacctatttt ttatgaatga atatttgcac tattactatt caaaactatt
     2401 aagatagcaa tcacattcaa tagccaaata ctataccacc tgagcgatgc aacgaaatga
     2461 tcaatttgag caaaaatgct gcatatttag gacggcatca ttatagaaat gcttcttgct
     2521 gtgtactttt ctctcgtctg gcagctgttt cgccgttatt gttaaaaccg gcttaagtta
     2581 ggtgtgtttt ctacgactag tgaatgccct actagaagat gtgtgttgca caaaatgtcc
     2641 ctggaataac caatttgaag tgcagatagc agtaaacgta agctaatatg aatattattt
     2701 aactgtaatg ttttaatatc gctggacatt actaataaac ccactataaa cacatgtaca
     2761 tatgtatgtt ttggcataca atgagtagtt ggggaaaaaa tgtgtaaaag caccgtgacc
     2821 atcacagcat aaagataacc agctgaagta tcgaatatga gtaaccccca aattgaatca
     2881 catgccgcaa ctgataggac ccatggaagt acactcttca tggcgatata caagacacac
     2941 acaagcacga acacccagtt gcggaggaaa ttctccgtaa atgaaaaccc aatcggcgaa
     3001 caattcatac ccatatatgg taaaagtttt gaacgcgact tgagagcgga gagcattgcg
     3061 gctgataagg ttttagcgct aagcgggctt tataaaacgg gctgcgggac cagttttcat
     3121 atcactaccg tttgagttct tgtgctgtgt ggatactcct cccgacacaa agccgctcca
     3181 tcagccagca gtcgtctaat ccagagaccc cggatcgggc tagcagtagg gtaccatgac
     3241 ttcgaaagtt tatgatccag aacaaaggaa acggatgata actggtccgc agtggtgggc
     3301 cagatgtaaa caaatgaatg ttcttgattc atttattaat tattatgatt cagaaaaaca
     3361 tgcagaaaat gctgttattt ttttacatgg taacgcggcc tcttcttatt tatggcgaca
     3421 tgttgtgcca catattgagc cagtagcgcg gtgtattata ccagacctta ttggtatggg
     3481 caaatcaggc aaatctggta atggttctta taggttactt gatcattaca aatatcttac
     3541 tgcatggttt gaacttctta atttaccaaa gaagatcatt tttgtcggcc atgattgggg
     3601 tgcttgtttg gcatttcatt atagctatga gcatcaagat aagatcaaag caatagttca
     3661 cgctgaaagt gtagtagatg tgattgaatc atgggatgaa tggcctgata ttgaagaaga
     3721 tattgcgttg atcaaatctg aagaaggaga aaaaatggtt ttggagaata acttcttcgt
     3781 ggaaaccatg ttgccatcaa aaatcatgag aaagttagaa ccagaagaat ttgcagcata
     3841 tcttgaacca ttcaaagaga aaggtgaagt tcgtcgtcca acattatcat ggcctcgtga
     3901 aatcccgtta gtaaaaggtg gtaaacctga cgttgtacaa attgttagga attataatgc
     3961 ttatctacgt gcaagtgatg atttaccaaa aatgtttatt gaatcggacc caggattctt
     4021 ttccaatgct attgttgaag gtgccaagaa gtttcctaat actgaatttg tcaaagtaaa
     4081 aggtcttcat ttttcgcaag aagatgcacc tgatgaaatg ggaaaatata tcaaatcgtt
     4141 cgttgagcga gttctcaaaa atgaacaata agaattctgc agatatccag cacagtggcg
     4201 gccgctcgag tctagactag aggcctgcgt cacgccactt caacgctcga tgggagcgtc
     4261 attggtgggc ggggtaaccg tcgaaatcag tgtttacgct tccaatcgca acaaaaaatt
     4321 cactgcaaca ctgaaaagca tacgaaaacg atgaagattg tacgagaaac cataaagtat
     4381 tttatccaca aagacacgta tagcagaaaa gccaagttaa ctcggcgata agttgtgtac
     4441 acaagaataa aatcggccag attcagtgtt gtcagaaata agaaaacccc actgtttttc
     4501 tttctgcagg ataccggtat aacgcgaaac agtggccagc ttttgttccc tttagtgagg
     4561 gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     4621 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     4681 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4741 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4801 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     4861 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4921 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4981 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     5041 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     5101 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     5161 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     5221 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     5281 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     5341 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5401 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     5461 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5521 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5581 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5641 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5701 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5761 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5821 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5881 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5941 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     6001 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     6061 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     6121 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     6181 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     6241 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     6301 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     6361 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     6421 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     6481 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     6541 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     6601 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     6661 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     6721 tccccgaaaa gtgccac
//
