LOCUS       pNTI733_Pol3_gRNA_[RPC3 7696 bp ds-DNA     circular     25-DEC-2020
DEFINITION  .
KEYWORDS    "marker:AmpR" "ref:pmid\:26376869"                                 
FEATURES             Location/Qualifiers
     protein_bind    6826..6859
                     /label="FRT (minimal)"
     misc_feature    280..686
                     /label="RPR1 promoter"
     CDS             complement(6373..6441)
                     /label="AmpR"
     rep_origin      complement(4869..5457)
                     /label="ori"
     misc_feature    complement(1161..2249)
                     /label="kl-Leu2 CDS"
     promoter        complement(6442..6533)
                     /label="AmpR promoter"
     protein_bind    604..622
                     /label="tet operator"
     misc_recomb     6826..6859
                     /label="FRT"
     promoter        complement(3373..3391)
                     /label="T7 promoter"
     misc_recomb     complement(4080..4113)
                     /label="FRT"
     misc_feature    258..272
                     /label="unstruc for gibson"
     primer_bind     complement(4583..4600)
                     /label="M13-fwd"
     primer_bind     complement(3401..3418)
                     /label="M13-fwd"
     misc_recomb     complement(3296..3329)
                     /label="LoxP"
     terminator      795..857
                     /label="RPR1 terminator"
     misc_feature    236..942
                     /label="gRNA expression cassette"
     CDS             complement(5581..6372)
                     /label="AmpR"
     protein_bind    complement(3296..3329)
                     /label="loxP"
     terminator      7502..7667
                     /label="ADH1 terminator"
     primer_bind     complement(3401..3417)
                     /label="M13 fwd"
     misc_RNA        707..782
                     /label="g RNA scaffold"
     terminator      30..219
                     /label="CYC1 terminator"
     misc_feature    236..257
                     /label="bcode"
     misc_recomb     complement(974..1007)
                     /label="LoxP"
     protein_bind    complement(4080..4113)
                     /label="FRT (minimal)"
     misc_feature    6900..7464
                     /label="Homology arm (X-3)"
     protein_bind    complement(974..1007)
                     /label="loxP"
     primer_bind     complement(3372..3392)
                     /label="T7"
     misc_feature    3493..4037
                     /label="homology arm (X-3)"
     primer_bind     complement(4583..4599)
                     /label="M13 fwd"
     primer          complement(707..744)
                     /label="509 R1 bc-gRNA R"
                     /note="sequence: TTCAGACGTGTGCTCTTCCGATCTagccttattttaacttgctatttctagctctaaaac"
ORIGIN
        1 caacggaatg cgtgcgatcg cgtgcattca tccgctctaa ccgaaaagga aggagttaga
       61 caacctgaag tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta
      121 tttatatttc aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata
      181 ctgaaaacct tgcttgagaa ggttttggga cgctcgaaga tcgcgtcagc tgaagAAGAC
      241 GTTATCGGGG CGCGCCTGAG ATCCAGTCAC TCGGGATCCg atctgccaat tgaacataac
      301 atggtagtta catatactag taatatggtt cggcacacat taaaagtata aaaactatct
      361 gaattacgaa ttacatatat tggtcataaa aatcaatcaa tcatcgtgtg ttttatatgt
      421 ctcttatcta agtataagaa tatccatagt taatattcac ttacgctacc ttttaacctg
      481 taatcattgt caacaggata tgttaacgac ccacattgat aaacgctagt atttcttttt
      541 cctcttctta ttggccggct gtctctatac tcccctatag tctgtttctt ttcgtttcga
      601 ttgtccctat cagtgataga gatggcgcac atggtacgct gtggtgctcg cggctgggaa
      661 cgaaactctg ggagctgcga ttggcaTTCT ATAGGTTGCT GCGATGgttt tagagctaga
      721 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
      781 gcttttttct cgagccatat ccaacttcca atttaatctt tcttttttaa ttttcactta
      841 tttgcgatac agaaagaaaa aagcgatagt aactattgaa ttttgtttgg atttggttag
      901 attagatatg gtttctcttt atatttacat gctaaaaatg ggcttcgtac gctgcaggtc
      961 gacaaccctt aatataactt cgtataatgt atgctatacg aagttattag gtctagagat
     1021 ccgcaggcta accggaacct gtattattta gtttatgcta cgttaaataa agacctttcg
     1081 ttcacataac tgaatgtgta atggccttga gatttcaagc ataccaagtt ggtggagacg
     1141 gggtcgttac aaaagactct ttaagccaag atttccttga cagccttggc gatagcatcg
     1201 ccaacctcag tggtagagtt agaaccacca aggtcaccgg ttctgacacc tgcatccaag
     1261 acatttctaa cagcttcttc aagagcccta ccttcttcaa ccaaatccaa ggataacttc
     1321 aacatcatag ctgcagataa gatggtagca attgggttaa ccttgtttgc tggtaaatct
     1381 ggggcagaac catgacatgg ttcgtacaaa ccgaatgcct tgttagtgtc aggtagggaa
     1441 gctagagatg cagaaggtaa taaacccaaa gaacctggaa taacagaggc ttcatcggag
     1501 ataatatcac caaacatgtt gttggtaata acaacaccgt ttagcttagt tggtgattta
     1561 accaaaatca tagcagcaga gtcgatcaat tggtgctgaa cagttaattg tgggaactca
     1621 gtcttgatgg tttcttcaac agtctttctc cacaatctgg aagaggcaag cacgttagcc
     1681 ttgtcaagtg accagattgg taatggtggg ttttgttgca atgccaagaa agcagccatt
     1741 cttgtaattc tttgaacttc aggaacactg tatttctcag agtcccaagc aactccgtca
     1801 ccttcatctt cttttctttc accaaagtag ataccaccaa ccaattctct aacgacgacg
     1861 aaatcggtac cctttgcata ttcaggcttc aaaggagaaa gatctagtaa agaatcagaa
     1921 gcaaagttac atggtcttaa gttggcgtat agacccaatt cctttctgat cttcaataga
     1981 ccttgttctg gtctaactgc gcccgtaccc cattttggac caccaacagc acctagtaag
     2041 acagcatcgg ctttcttaga ggcttctaga gcttcatctg gtaaaggagt gccagtggca
     2101 tcgatggcag cacccccgat caagtgatgt tggaaattga acttaatttc tggacggact
     2161 tcagcaatgg cattcaagac cttaatagct tcgtcagtaa cttctttacc gacgtgatca
     2221 cccggtagga caacgatatt cttagacatg gtataatctg tgtagtgtgg gatacttttt
     2281 acttcttcaa ataggtatca acaataaaaa taaatcgaag caaatgtagg aatgcgttaa
     2341 agcagatgta cttacttaga gtacatagat atatttatat aattcaatat ataaaagtat
     2401 atgaagcatc tgatgttgaa cctgtcatga ctctaaactg gaatgggtag ttatgcttct
     2461 gaaggttttc cgttagaaaa tgagtgcagg gatcaggccc ggaaccggtt ttagcctgaa
     2521 aggaaaagaa aaaaatgcgg tgattactag cacgtgactg cgctgaattg gaatccacca
     2581 caagtacagc gaatggcaag agggaagggg aatatattgt gcctggcggt agtctgctat
     2641 atcaaatgtt aaacataact aaatgcagta gtgaatagag taatgtattt ctacatatgt
     2701 gggtagggcc aatgggagcc cgatgtctat tgtacagata ttttcctttt atagttaatc
     2761 aattcaactt ggaaatcatt gagtcgatat ctacttggat aactggagcc gtggcagcgt
     2821 ttgaaatttg acgagtctcc ttgttttgtt cgcgcatttt cagtttattc aaacgggaga
     2881 tggcaccgaa tccactgact tctctgttca atttgaatgg taataggaag aaagtacatt
     2941 tcgagaaatt ttcgcccaac gtcgaaatct catggatgat atcctcgata caaatgacac
     3001 cttcatcacc taatttctct tcaacgatat tgttgtcatt caagattatc tcttttggtt
     3061 ctttatcttc tggcctttgc cacattattc tagatctctt ttggattaaa gaacggatag
     3121 atgcaagtga aggttgtccg ataacaatat atggtgatac aagtttcaac aatgggtaca
     3181 cgtccttggt aagtttgatg aaaacaccag tgttcaactc aacaagtctc aaaagagata
     3241 acactttgta agcctttgct gggatcttca cagcgagctc tcgagaaccc ttaatataac
     3301 ttcgtataat gtatgctata cgaagttatt aggtgatatc agatccacta gtggcctatg
     3361 cacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
     3421 gtcgtgactg ggaaaaccct ggcgttaccc ctgcaggact agtgctgagg cattaattgt
     3481 gtccgcgttt ctaaggccac ttttcaatga aacggatatt gatatgctag taaaaggacg
     3541 agctcaagag cgaaaatata agtaaagaat tcgagtgcac ttgtctccat gcagcaagat
     3601 ttcatatgag tcttttttat ctttttactt tttacattac acgatatgca ctttatgaaa
     3661 atttaacgag gttggaagcc ggataatcaa ccaaaatcag gcacgaaggc acactcgtat
     3721 atgcatgttg ttgaaactct gttacgctga actaacaatc acacatgtag aggtcaccgg
     3781 gaaaagttgc gaccccatgg aaggtcgatc tcttcgtttg gctttgcttg gctggcggca
     3841 ttgcgcttct tcgcttatac ccgtctcttg acgctcgagc tcgttcattg agataccttt
     3901 attcttgcac attttctggc ttttttcgct actcgggtac atgtaatcat gcacacagaa
     3961 ggtgctgtag ggtgaaagtt cctttgtgct gtcgtttgtt tttaatgcca aactttccgg
     4021 tgatcaataa ccacctcatt aagacctcag cgcggccgca aatttaaata aaatgaagtg
     4081 aagttcctat actttctaga gaataggaac ttctatagtg agtcgaataa gggcgacaca
     4141 aaatttattc taaatgcata ataaatactg ataacatctt atagtttgta ttatattttg
     4201 tattatcgtt gacatgtata attttgatat caaaaactga ttttcccttt attattttcg
     4261 agatttattt tcttaattct ctttaacaaa ctagaaatat tgtatataca aaaaatcata
     4321 aataatagat gaatagttta attataggtg ttcatcaatc gaaaaagcaa cgtatcttat
     4381 ttaaagtgcg ttgctttttt ctcatttata aggttaaata attctcatat atcaagcaaa
     4441 gtgacaggcg cccttaaata ttctgacaaa tgctctttcc ctaaactccc cccataaaaa
     4501 aacccgccga agcgggtttt tacgttattt gcggattaac gattactcgt tatcagaacc
     4561 gcccaggggg cccgagctta agactggccg tcgttttaca acacagaaag agtttgtaga
     4621 aacgcaaaaa ggccatccgt caggggcctt ctgcttagtt tgatgcctgg cagttcccta
     4681 ctctcgcctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4741 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4801 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4861 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4921 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     4981 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     5041 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     5101 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     5161 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5221 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5281 tggtgggcta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     5341 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     5401 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     5461 gatcctttga tcttttctac ggggtctgac gctcagtgga acgacgcgcg cgtaactcac
     5521 gttaagggat tttggtcatg agcttgcgcc gtcccgtcaa gtcagcgtaa tgctctgctt
     5581 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5641 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5701 cagcgctgcg atgataccgc gagaaccacg ctcaccggct ccggatttat cagcaataaa
     5761 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5821 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     5881 cgttgttgcc atcgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     5941 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6001 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6061 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6121 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6181 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6241 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6301 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6361 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6421 acggaaatgt tgaatactca tattcttcct ttttcaatat tattgaagca tttatcaggg
     6481 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6541 cagtgttaca accaattaac caattctgaa cattatcgcg agcccattta tacctgaata
     6601 tggctcataa caccccttgt ttgcctggcg gcagtagcgc ggtggtccca cctgacccca
     6661 tgccgaactc agaagtgaaa cgccgtagcg ccgatggtag tgtggggact ccccatgcga
     6721 gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt
     6781 cgcccgggct aattatgggg tgtcgccctt attcgactct atagtgaagt tcctattctc
     6841 tagaaagtat aggaacttct gaagtgggga tttaaatgcg gccgcgctga gggtttaatc
     6901 gagatctttg tgttcggtta cccggctcag atcctaactt ggtatgttta ttcgtataag
     6961 ttactgttgt ccacaggcaa tactctgcag aaaattaaaa cggcattaat gctaggacaa
     7021 ccagaattgt tactactgta tgtgcgatag ttgataactg caacattatg cccggtatat
     7081 tctcaaaaaa ccctattact gcatacgaag aaatcgcaag agaaatcttt cggtttggaa
     7141 aagctcactg tgaggttcct tggagccaat agtaatacag cacaatccaa ggaaaaatct
     7201 ggcctatatg caaggaagga gagatagtca aaagcattct ttcccctaga agttggtgca
     7261 tatatggcat cgttaaaaca tattaccccc aaaatttctt ctctaaacga tgtgcttggc
     7321 ctttgttttg gtttttgatg tcggtcgttt gaggcccctt gcggaaaatc gagatcgccg
     7381 aatggcacgc gagggaaggg aaataaggtt taaaggcact gaaacaatag gcaagaagta
     7441 ggcgagagcc gacatacgag actattaagt cctcagcgag ctcgcatgga atgcgtgcga
     7501 tgagcgacct catgctatac ctgagaaagc aacctgacct acaggaaaga gttactcaag
     7561 aataagaatt ttcgttttaa aacctaagag tcactttaaa atttgtatac acttattttt
     7621 tttataactt atttaataat aaaaatcata aatcataaga aattcgctta tttagaagtg
     7681 tcaacaacgt atctac
//