LOCUS       62056_10905        9460 bp DNA     circular SYN 01-FEB-2020
DEFINITION  Cloning vector pFLHD, complete sequence.
ACCESSION   MN043927
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9460)
  AUTHORS   Gunadi A, Finer J.
  TITLE     CRISPRi validation of regulatory elements in a soybean (Glycine max)
            elongation factor-1 alpha promoter
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9460)
  AUTHORS   Gunadi A, Finer J.
  TITLE     Direct Submission
  JOURNAL   Submitted (06-JUN-2019) Horticulture and Crop Science, The Ohio 
            State University, 216 Williams Hall, 1680 Madison Ave., Wooster, OH 
            44691, USA
REFERENCE   3  (bases 1 to 9460)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (06-JUN-2019) Horticulture and Crop Science, The Ohio State 
            University, 216 Williams Hall, 1680 Madison Ave., Wooster, OH 44691,
            USA"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..9460
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      1..253
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(295..311)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(319..335)
                     /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(343..373)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(388..409)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(697..1285)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1459..2316)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(2317..2421)
                     /label=AmpR promoter
     regulatory      2745..5169
                     /label=GmubiXL promoter from soybean
                     /note="GmubiXL promoter from soybean"
                     /regulatory_class="promoter"
     CDS             5303..9403
                     /label=dCas9
                     /note="catalytically dead mutant of the Cas9 endonuclease
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
ORIGIN
        1 gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg
       61 atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc
      121 atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac
      181 gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct
      241 atgttactag atccgatgat aagctgtcaa acatgagaat tcggcgtaat catggtcata
      301 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
      361 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
      421 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
      481 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
      541 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
      601 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
      661 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
      721 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
      781 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
      841 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
      901 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
      961 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     1021 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     1081 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     1141 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     1201 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     1261 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     1321 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     1381 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     1441 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     1501 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     1561 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     1621 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     1681 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     1741 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     1801 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     1861 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     1921 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     1981 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     2041 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     2101 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     2161 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     2221 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     2281 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     2341 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     2401 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     2461 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc
     2521 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     2581 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     2641 cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca
     2701 tatccagggt tttcccagtc acgacgtcga cggcccgggc tggtacataa gtctatctaa
     2761 aatggaaaga ttgttaccta atctcaaatc atatttttcc catcataaaa aaaaatctca
     2821 aatcatgtga tcttgtcgat catcatttaa tacattatta attcttcttt ttaattgaaa
     2881 aatgtgatta tatgttatgt ggagattagt tcagaccatg atccttgaat atgtagtaag
     2941 tttttgtaat gttgagtttg agttctttga cactaggtcc atgtttccca aaattaagtg
     3001 taaaaaataa ataaagaatt aaaagttgtg aaaaaaattg tcctcgttta tttttattat
     3061 tcagaattaa tctttattga gactaaaatt atttacagaa agttaataca gcactaaaag
     3121 ttattttatt tttttgagaa acaaaaactg aaatctaaaa aaaaataaga aaccggaaat
     3181 ctaattaaca aaaatatata cgggctaaca tggttatggg cttaggctgc ctcattaccc
     3241 attacatgct ctgttcctaa aaaaaaaaag aagtttgggc ctttatcctc cagtaagttt
     3301 tttaattggg ctcaagaact ttaggattta ggtgaggatc aatgaaagtt tatatattag
     3361 gcttgatttt tttttcaatt tacgtaaaaa tgaaagaaaa agtcacactc tataaacttt
     3421 atttttttca atattaatac attgttattg tggatatatc attagcggaa aagtaattct
     3481 aataccagat ttaattgact ttgactaatt tttttagcgg aataaaagtt ttgntacttt
     3541 gncgcgtgat ttttaatnna actttgggga taatttcccc tccaattcag cccaaaaaaa
     3601 aaaaaactga nnncatatnt tattagtagg caacttgttc gtaaatggtg tggcttatcc
     3661 gaatggaaat nggattcgtt ttctttaaca tcattattgt ttttgtcaat gagctatctt
     3721 ttagtcttat gttattggtg aatctgtcct taagttgcat catttaacac atctcctcat
     3781 tagagaaaaa aattcttccc taaacgattg gtagtaaaaa catctaataa gaaataagaa
     3841 agaaaaatta ggaaaaagga aagttcatta aaaaaaatat tttgaattat tttttaaaaa
     3901 atatctaaat attttttaaa tgaataattt tatataaact gtaactaaat gtatacaagt
     3961 aatgtatgtt aaaaaaatac ttgaaaaatc tactgaaaat atatcttaca aggtgaaatt
     4021 aaataagaaa gaatttagtg gaataattat gattttattt aaaaaataat tattaaagat
     4081 ttttttgctc cataataaga aaacttttca attattcttt tctggtccat aataaaaaaa
     4141 atctagcatg acagcttttc catagatttt taataatgta aaagcagccg acttcaggca
     4201 atggatagtg gggcccgtat caacttcgga cgctccactt gcaacggggt gggcccaata
     4261 taacaacgac gtcgtaacag ataaagcgaa gcttgaaggt gcatgtgact ccgtcaagat
     4321 tacgaaaccg ccaactacca cgcaaattgc aattctcaat ttcctagaag gactctccga
     4381 aaatgcatcc aataccaaat attacccgtg tcataggcac caagtgacac catacatgaa
     4441 cacgcgtcac aatatgactg gagaagggtt ccacacctta tgctataaaa cgccccacac
     4501 ccctcctcct tccttcgcag ttcaattcca atatattcca ttctctctgt gtatttccct
     4561 acctctccct tcaaggttag tcgatttctt ctgtttttct tcttcgttct ttccatgaat
     4621 tgtgtatgtt ctttgatcaa tacgatgttg atttgattgt gttttgtttg gtttcatcga
     4681 tcttcaattt tcataatcag attcagcttt tattatcttt acaacaacgt ccttaatttg
     4741 atgattcttt aatcgtagat ttgctctaat tagagctttt tcatgtcaga tccctttaca
     4801 acaagcctta attgttgatt cattaatcgt agattagggc ttttttcatt gattacttca
     4861 gatccgttaa acgtaaccat agatcagggc tttttcatga attacttcag atccgttaaa
     4921 caacagcctt attttttata cttctgtggt ttttcaagaa attgttcaga tccgttgaca
     4981 aaaagcctta ttcgttgatt ctatatcgtt tttcgagaga tattgctcag atctgttagc
     5041 aactgccttg tttgttgatt ctattgccgt ggattagggt tttttttcac gagattgctt
     5101 cagatccgta cttaagatta cgtaatggat tttgattctg atttatctgt gattgttgac
     5161 tcgacaggag cggccgctct agatggatta caaggaccac gacggggatt acaaggacca
     5221 cgacattgat tacaaggatg atgatgacaa gatggctccg aagaagaaga ggaaggttgg
     5281 catccacggg gtgccagctg ctgacaagaa gtactcgatc ggcctcgcca ttgggactaa
     5341 ctctgttggc tgggccgtga tcaccgacga gtacaaggtg ccctcaaaga agttcaaggt
     5401 cctgggcaac accgatcggc attccatcaa gaagaatctc attggcgctc tcctgttcga
     5461 cagcggcgag acggctgagg ctacgcggct caagcgcacc gcccgcaggc ggtacacgcg
     5521 caggaagaat cgcatctgct acctgcagga gattttctcc aacgagatgg cgaaggttga
     5581 cgattctttc ttccacaggc tggaggagtc attcctcgtg gaggaggata agaagcacga
     5641 gcggcatcca atcttcggca acattgtcga cgaggttgcc taccacgaga agtaccctac
     5701 gatctaccat ctgcggaaga agctcgtgga ctccacagat aaggcggacc tccgcctgat
     5761 ctacctcgct ctggcccaca tgattaagtt caggggccat ttcctgatcg agggggatct
     5821 caacccggac aatagcgatg ttgacaagct gttcatccag ctcgtgcaga cgtacaacca
     5881 gctcttcgag gagaacccca ttaatgcgtc aggcgtcgac gcgaaggcta tcctgtccgc
     5941 taggctctcg aagtctcggc gcctcgagaa cctgatcgcc cagctgccgg gcgagaagaa
     6001 gaacggcctg ttcgggaatc tcattgcgct cagcctgggg ctcacgccca acttcaagtc
     6061 gaatttcgat ctcgctgagg acgccaagct gcagctctcc aaggacacat acgacgatga
     6121 cctggataac ctcctggccc agatcggcga tcagtacgcg gacctgttcc tcgctgccaa
     6181 gaatctgtcg gacgccatcc tcctgtctga tattctcagg gtgaacaccg agattacgaa
     6241 ggctccgctc tcagcctcca tgatcaagcg ctacgacgag caccatcagg atctgaccct
     6301 cctgaaggcg ctggtcaggc agcagctccc cgagaagtac aaggagatct tcttcgatca
     6361 gtcgaagaac ggctacgctg ggtacattga cggcggggcc tctcaggagg agttctacaa
     6421 gttcatcaag ccgattctgg agaagatgga cggcacggag gagctgctgg tgaagctcaa
     6481 tcgcgaggac ctcctgagga agcagcggac attcgataac ggcagcatcc cacaccagat
     6541 tcatctcggg gagctgcacg ctatcctgag gaggcaggag gacttctacc ctttcctcaa
     6601 ggataaccgc gagaagatcg agaagattct gactttcagg atcccgtact acgtcggccc
     6661 actcgctagg ggcaactccc gcttcgcttg gatgacccgc aagtcagagg agacgatcac
     6721 gccgtggaac ttcgaggagg tggtcgacaa gggcgctagc gctcagtcgt tcatcgagag
     6781 gatgacgaat ttcgacaaga acctgccaaa tgagaaggtg ctccctaagc actcgctcct
     6841 gtacgagtac ttcacagtct acaacgagct gactaaggtg aagtatgtga ccgagggcat
     6901 gaggaagccg gctttcctgt ctggggagca gaagaaggcc atcgtggacc tcctgttcaa
     6961 gaccaaccgg aaggtcacgg ttaagcagct caaggaggac tacttcaaga agattgagtg
     7021 cttcgattcg gtcgagatct ctggcgttga ggaccgcttc aacgcctccc tggggaccta
     7081 ccacgatctc ctgaagatca ttaaggataa ggacttcctg gacaacgagg agaatgagga
     7141 tatcctcgag gacattgtgc tgacactcac tctgttcgag gaccgggaga tgatcgagga
     7201 gcgcctgaag acttacgccc atctcttcga tgacaaggtc atgaagcagc tcaagaggag
     7261 gaggtacacc ggctggggga ggctgagcag gaagctcatc aacggcattc gggacaagca
     7321 gtccgggaag acgatcctcg acttcctgaa gagcgatggc ttcgcgaacc gcaatttcat
     7381 gcagctgatt cacgatgaca gcctcacatt caaggaggat atccagaagg ctcaggtgag
     7441 cggccagggg gactcgctgc acgagcatat cgcgaacctc gctggctcgc cagctatcaa
     7501 gaaggggatt ctgcagaccg tgaaggttgt ggacgagctg gtgaaggtca tgggcaggca
     7561 caagcctgag aacatcgtca ttgagatggc ccgggagaat cagaccacgc agaagggcca
     7621 gaagaactca cgcgagagga tgaagaggat cgaggagggc attaaggagc tggggtccca
     7681 gatcctcaag gagcacccgg tggagaacac gcagctgcag aatgagaagc tctacctgta
     7741 ctacctccag aatggccgcg atatgtatgt ggaccaggag ctggatatta acaggctcag
     7801 cgattacgac gtcgatgcca tcgttccaca gtcattcctg aaggatgact ccattgacaa
     7861 caaggtcctc accaggtcgg acaagaaccg gggcaagtct gataatgttc cttcagagga
     7921 ggtcgttaag aagatgaaga actactggcg ccagctcctg aatgccaagc tgatcacgca
     7981 gcggaagttc gataacctca caaaggctga gaggggcggg ctctctgagc tggacaaggc
     8041 gggcttcatc aagaggcagc tggtcgagac acggcagatc actaagcacg ttgcgcagat
     8101 tctcgactca cggatgaaca ctaagtacga tgagaatgac aagctgatcc gcgaggtgaa
     8161 ggtcatcacc ctgaagtcaa agctcgtctc cgacttcagg aaggatttcc agttctacaa
     8221 ggttcgggag atcaacaatt accaccatgc ccatgacgcg tacctgaacg cggtggtcgg
     8281 cacagctctg atcaagaagt acccaaagct cgagagcgag ttcgtgtacg gggactacaa
     8341 ggtttacgat gtgaggaaga tgatcgccaa gtcggagcag gagattggca aggctaccgc
     8401 caagtacttc ttctactcta acattatgaa tttcttcaag acagagatca ctctggccaa
     8461 tggcgagatc cggaagcgcc ccctcatcga gacgaacggc gagacggggg agatcgtgtg
     8521 ggacaagggc agggatttcg cgaccgtcag gaaggttctc tccatgccac aagtgaatat
     8581 cgtcaagaag acagaggtcc agactggcgg gttctctaag gagtcaattc tgcctaagcg
     8641 gaacagcgac aagctcatcg cccgcaagaa ggactgggat ccgaagaagt acggcgggtt
     8701 cgacagcccc actgtggcct actcggtcct ggttgtggcg aaggttgaga agggcaagtc
     8761 caagaagctc aagagcgtga aggagctgct ggggatcacg attatggagc gctccagctt
     8821 cgagaagaac ccgatcgatt tcctggaggc gaagggctac aaggaggtga agaaggacct
     8881 gatcattaag ctccccaagt actcactctt cgagctggag aacggcagga agcggatgct
     8941 ggcttccgct ggcgagctgc agaaggggaa cgagctggct ctgccgtcca agtatgtgaa
     9001 cttcctctac ctggcctccc actacgagaa gctcaagggc agccccgagg acaacgagca
     9061 gaagcagctg ttcgtcgagc agcacaagca ttacctcgac gagatcattg agcagatttc
     9121 cgagttctcc aagcgcgtga tcctggccga cgcgaatctg gataaggtcc tctccgcgta
     9181 caacaagcac cgcgacaagc caatcaggga gcaggctgag aatatcattc atctcttcac
     9241 cctgacgaac ctcggcgccc ctgctgcttt caagtacttc gacacaacta tcgatcgcaa
     9301 gaggtacaca agcactaagg aggtcctgga cgcgaccctc atccaccagt cgattaccgg
     9361 cctctacgag acgcgcatcg acctgtctca gctcgggggc gacaagcggc cagcggcgac
     9421 gaagaaggcg gggcaggcga agaagaagaa gtgaccatgg
//
