LOCUS       62056_10850        3661 bp DNA     circular SYN 01-FEB-2020
DEFINITION  Cloning vector pFLGA-4xGbox-HITI-1, complete sequence.
ACCESSION   MN043935
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3661)
  AUTHORS   Gunadi A, Orchard N, Qu F, Finer J.
  TITLE     Enhanced CRISPR/Cas9 mediated homology-independent targeted 
            integration in plant cotyledonary tissues
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3661)
  AUTHORS   Gunadi A, Orchard N, Qu F, 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 3661)
  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            "Unpublished"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..3661
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(4..20)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        494..598
                     /label=AmpR promoter
     CDS             599..1456
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      1630..2218
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    2506..2527
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        2542..2572
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2580..2596
                     /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)."
     primer_bind     2604..2620
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   2718..2895
                     /label=4x EF4 containing G-box motifs
                     /rpt_family="regulatory motif"
                     /note="4x EF4 containing G-box motifs"
     regulatory      2928..3279
                     /label=U6.6 Promoter from Medicago truncatula
                     /note="U6.6 Promoter from Medicago truncatula"
                     /regulatory_class="promoter"
     tRNA            3287..3361
                     /product="tRNA-Gly"
                     /label=plant tRNA (Gly)
                     /note="plant tRNA (Gly)"
     misc_RNA        3383..3458
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      3459..3650
                     /label=U6-26 pol III terminator from Arabidopsis thaliana
                     /note="U6-26 pol III terminator from Arabidopsis thaliana"
                     /regulatory_class="terminator"
ORIGIN
        1 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
       61 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
      121 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct
      181 ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc
      241 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
      301 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
      361 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
      421 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
      481 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
      541 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
      601 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
      661 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
      721 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
      781 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
      841 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
      901 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
      961 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     1021 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     1081 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     1141 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     1201 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     1261 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtggttctcg
     1321 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     1381 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     1441 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     1501 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     1561 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     1621 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     1681 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     1741 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     1801 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     1861 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     1921 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     1981 gcgaacgacc tacaccgaac tgagatacct acagcgtgag cattgagaaa gcgccacgct
     2041 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     2101 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     2161 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     2221 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     2281 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     2341 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     2401 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     2461 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     2521 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     2581 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttat
     2641 taccctgtta tccctagata tcgattttgg ctaccttaac tctcttaagg tagcctgcag
     2701 ctagcggtac cactagtgtt ccccgtgaaa gtgacacgtg gcaggacttg ggacgtgtct
     2761 agtgttcccc gtgaaagtga cacgtggcag gacttgggac gtgtctagtg ttccccgtga
     2821 aagtgacacg tggcaggact tgggacgtgt ctagtgttcc ccgtgaaagt gacacgtggc
     2881 aggacttggg acgtgtctag agtcgaccgc aagacccttc ctctataatg cctatcttat
     2941 atgatcaatg aggcatttaa ttgggtgcat atgatggtga aaaaaggtgc agctcctggc
     3001 ttgggaatga tgactcatgt ggaatttggt cttaaattta tcacatcctt ttgggatgtg
     3061 atgattgtat cacttgttca ttttgcaaag acaaggtgca ctgctacaaa ctttggttta
     3121 atctgaaata aaacaaaact cactgagagg aagatgcatc ccagtaggtg aaagtcgaga
     3181 aggatttgca tgttactatt acacttgctt tttagtccca catcgtctga aacataaaat
     3241 atttcagcgt ttaaatactt caagcgaacc agtaggcttc ctaggaacaa agcaccagtg
     3301 gtctagtggt agaatagtac cctgccacgg tacagacccg ggttctattc ccggctggtg
     3361 caatttagct gaagcaaagc aagttttaga gctagaaata gcaagttaaa ataaggctag
     3421 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttttgcaa aattttccag
     3481 atcgatttct tcttcctctg ttcttcggcg ttcaatttct ggggttttct cttcgttttc
     3541 tgtaactgaa acctaaaatt tgacctaaaa aaaatctcaa ataatatgat tcagtggttt
     3601 tgtacttttc agttagttga gttttgcagt tccgatgaga taaaccaata agcttgcatg
     3661 c
//
