LOCUS       pEP0001_pIVTRup(BsmBI)_EGFP        4926 bp    DNA     circular UNA 01-JUL-2019
DEFINITION  Concatenation of 2 sequences.
ACCESSION   urn.local...3i-br7smtl
VERSION     urn.local...3i-br7smtl
KEYWORDS    .
SOURCE      
  ORGANISM  
            .
FEATURES             Location/Qualifiers
     misc_feature    1..4155
                     /Source="referenceToDocument: urn:local:.:s7-br7sn3o"
                     /label="PCR_01_Backbone Copy for Gibson  (concatenated
                     sequence 1)"
                     /note="Geneious type: Concatenated sequence"
     misc_feature    0^1
                     /Original_Bases="ATCGCTGCCTTCTGCGGGGCATCGCTGCCTTCTGCGGGGCT
                     TGCCT"
                     /label="ATCGCTGCCTTCTGCGGGGCATCGCTGCCTTCTGCGGGGCTTGCCT"
                     /note="Geneious type: Editing History Deletion"
     cds             <1..81
                     /created_by="User"
                     /modified_by="User"
                     /X-Transferred_From="101362 - pIVTRup Copy; dCas9-VP64"
                     /X-Transferred_Similarity="100.00%"
                     /Transferred_From="101362 - pIVTRup Copy, 101362 - pIVTRup
                     PCR DraI, Digestion of 101362 - pIVTRup PCR DraI -
                     Fragment 3, dCas9-VP64"
                     /Transferred_Similarity="100.00%"
                     /label="5'UTR"
     misc_feature    complement(59..81)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="12899990249898143dbe7-3773-427a-aa05-1a
                     94cee5f9f8"
                     /%GC="52.2"
                     /Tm="58.8"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CTAGACCCTCACTTCCTACTCAG"
                     /label="pIVTRup_BsmBI_insert_REV"
                     /note="Geneious type: primer_bind_reverse"
     3'UTR           82..136
                     /created_by="User"
                     /label="polyA(55)"
     misc_feature    complement(138..143)
                     /Recognition_pattern="CGTCTC(1/5)"
                     /annotation_group="CGTCTC"
                     /label="BsmBI"
                     /note="Geneious type: restriction site"
     protein_bind    282..298
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
                     /label="lac operator"
     promoter        complement(306..336)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    351..372
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                      of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(660..1248)
                     /direction="LEFT"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /label="ori"
     rep_origin      complement(3435..3863)
                     /direction="LEFT"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     primer_bind     3954..3973
                     /Mismatches=0
                     /%GC="50.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Tm="58.8"
                     /annotation_group="4857420885142100418b4092-1ee7-41a8-9f35
                     -cafb4a644926"
                     /Sequence="TGCAAGGCGATTAAGTTGGG"
                     /created_by="primer3"
                     /label="PCR_0128"
     promoter        4027..4045
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /label="T7 promoter"
     primer_bind     4038..4056
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="12899407903902e50d4d7-3dfd-4a1d-9a48-af
                     2709fb6efc"
                     /%GC="52.6"
                     /Tm="57.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="5.4"
                     /Sequence="ACTATAGGGCGAATTGGGC"
                     /label="pIVTRup_BsmBI_insert_FWD"
     promoter        4117..4135
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /label="T7 promoter"
     cds             4134..>4155
                     /created_by="User"
                     /modified_by="User"
                     /X-Transferred_From="101362 - pIVTRup Copy"
                     /X-Transferred_Similarity="100.00%"
                     /Transferred_From="101362 - pIVTRup Copy, 101362 - pIVTRup
                     PCR DraI, Digestion of 101362 - pIVTRup PCR DraI -
                     Fragment 3"
                     /Transferred_Similarity="100.00%"
                     /label="3'UTR"
     misc_feature    4156..4926
                     /Source="referenceToDocument: urn:local:.:s8-br7sn3o"
                     /label="PCR_02_EGFP (concatenated sequence 2)"
                     /note="Geneious type: Concatenated sequence"
     misc_feature    4155^4156
                     /Original_Bases="AGTAAGAAGAAATATAAGAGCCACC"
                     /label="AGTAAGAAGAAATATAAGAGCCACC"
                     /note="Geneious type: Editing History Deletion"
     primer_bind     4181..4201
                     /Mismatches=0
                     /Extension="AGTAAGAAGAAATATAAGAGCCACC"
                     /created_by="primer3"
                     /annotation_group="864183859804110a4180c-1856-45ef-bef0-48
                     1c03dc207f"
                     /%GC="61.9"
                     /Tm="64.5"
                     /Hairpin_Tm="47.5"
                     /Self_Dimer_Tm="None"
                     /Sequence="ATGGTGAGCAAGGGCGAGGAG"
                     /label="EGFP_FWD"
     CDS             4181..4897
                     /codon_start=1
                     /product="enhanced GFP"
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFK
                     DDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKN
                     GIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDH
                     MVLLEFVTAAGITLGMDELYK"
                     /label="EGFP"
     misc_feature    complement(4874..4897)
                     /Mismatches=0
                     /Extension="AGGCAAGCCCCGCAGAAGGCAGCGATTCA"
                     /created_by="primer3"
                     /annotation_group="86418970886819559e3b8-8303-4568-9356-98
                     1cb2e1cb13"
                     /%GC="58.3"
                     /Tm="65.5"
                     /Hairpin_Tm="50.9"
                     /Self_Dimer_Tm="21.8"
                     /Sequence="CTTGTACAGCTCGTCCATGCCGAG"
                     /label="EGFP_REV"
                     /note="Geneious type: primer_bind_reverse"
ORIGIN      
        1 tctggccatg cccttcttct ctcccttgca cctgtacctc ttggtctttg aataaagcct
       61 gagtaggaag tgagggtcta gaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
      121 aaaaaaaaaa aaaaaatgag acgcgatcaa taaccgacac tcctgcgtcg cctacgcgct
      181 acgatctgca gaattccagc acactggcgg ccgttactag tggatccgag ctcggtacca
      241 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
      301 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
      361 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
      421 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
      481 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
      541 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
      601 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
      661 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
      721 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
      781 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
      841 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
      901 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
      961 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     1021 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     1081 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     1141 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     1201 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     1261 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     1321 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     1381 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     1441 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     1501 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     1561 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     1621 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     1681 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     1741 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     1801 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     1861 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     1921 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     1981 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     2041 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     2101 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     2161 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     2221 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     2281 ctcttccttt ttcaattcag aagaactcgt caagaaggcg atagaaggcg atgcgctgcg
     2341 aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg ccgccaagct
     2401 cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc acacccagcc
     2461 ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc ggcaagcagg
     2521 catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg agcctggcga
     2581 acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac
     2641 cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc
     2701 aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct
     2761 cggcaggagc aaggtgggat gacaggagat cctgccccgg cacttcgccc aatagcagcc
     2821 agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg
     2881 ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg
     2941 tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc
     3001 agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag
     3061 aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct gtctcttgat
     3121 cagatcttga tcccctgcgc catcagatcc ttggcggcaa gaaagccatc cagtttactt
     3181 tgcagggctt cccaacctta ccagagggcg ccccagctgg caattccggt tcgcttgctg
     3241 tccataaaac cgcccagtct agctatcgcc atgtaagccc actgcaagct acctgctttc
     3301 tctttgcgct tgcgttttcc cttgtccaga tagcccagta gctgacattc atccggggtc
     3361 agcaccgttt ctgcggactg gctttctacg tgttccgctt cctttagcag cccttgcgcc
     3421 ctgaattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     3481 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     3541 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     3601 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     3661 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     3721 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     3781 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     3841 taatgcgccg ctacagggcg cgtccattcg ccattcaggc tgcgcaactg ttgggaaggg
     3901 cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg
     3961 cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt
     4021 gaattgtaat acgactcact atagggcgaa ttgggccctc tagatgcatg ctcgagcggc
     4081 cgccagtgtg atggatttgg accctcgtac agaagctaat acgactcact atagggaaat
     4141 aagagagaaa agaagagtaa gaagaaatat aagagccacc atggtgagca agggcgagga
     4201 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     4261 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     4321 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     4381 tggagtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     4441 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     4501 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     4561 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     4621 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     4681 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     4741 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     4801 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     4861 cgccgggatc actctcggca tggacgagct gtacaagtga atcgctgcct tctgcggggc
     4921 ttgcct
//
