LOCUS       pGMF2-M                 4101 bp    DNA     circular SYN 09-JUL-2025
DEFINITION  Level 1 OsU6-2p::sgRNA2.
ACCESSION   urn.local...q6-je0751q
KEYWORDS    .
SOURCE      Integrated DNA Technologies
  ORGANISM  .
FEATURES             Location/Qualifiers
     Misc._feature   520..667
                     /vntifkey=21
                     /note="Unknown feature type:Misc._feature"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="pICSL11059 35Sp::HPTII:35St"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; LB (<a
                     href=""urn:local:.:7jr-d1yz7zq"">pICSL11059
                     35Sp::HPTII:35St</a>)"
                     /Alternative_Match="100.00%; Adjusted-Interval-0:0/0; LB
                     (<a href=""urn:local:.:w-c2ilwp9"">pFH52</a>)"
                     /Alternative_Match="100.00%; Adjusted-Interval-0:0/0; LB
                     (<a href=""urn:local:.:f-c2iltpe"">pICSL11059</a>)"
                     /Alternative_Match="100.00%; Adjusted-Interval-0:0/0; LB
                     (<a href=""urn:local:.:7jz-d1yz8pq"">pFH52
                     2x35Sp::SpCas9:NOSt</a>)"
                     /label="LB"
     primer_bind     586..609
                     /%GC="50.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Tm="61.3"
                     /annotation_group="1214696317113300e06d0f4e-a2e5-421b-a346
                     -887336be90c4"
                     /created_by="primer3"
                     /Sequence="CTGGTGGCAGGATATATTGTGGTG"
                     /Mismatches=0
                     /#_Local_Off-target_Sites=0
                     /Local_Off-target="pGMF2-M"
                     /label="GMF002"
     promoter        723..969
                     /created_by="Florian"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="L1_target2 in pTwist Amp High Copy -
                     fake primer PDS target2"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; OsU6-2
                     promoter (<a href=""urn:local:.:5d-c2iy4z2"">L1_target2 in
                     pTwist Amp High Copy - fake primer PDS target2</a>)"
                     /Alternative_Match="100.00%; Adjusted-Interval-0:0/0;
                     OsU6-2 promoter (<a
                     href=""urn:local:.:1f-c2incxl"">L1_target2 in pTwist Amp
                     High Copy</a>)"
                     /Alternative_Match="100.00%; Adjusted-Interval-0:0/0;
                     OsU6-2 promoter (<a
                     href=""urn:local:.:1b-c2incxi"">L1_target1 in pTwist Amp
                     High Copy</a>)"
                     /Alternative_Match="100.00%; Adjusted-Interval-0:0/0;
                     OsU6-2 promoter (<a href=""urn:local:.:60-c2iy93f"">Level2
                     Plantain PDS KO vector final assembly</a>)"
                     /Alternative_Match="100.00%; Adjusted-Interval-0:0/0;
                     OsU6-2 promoter (<a href=""urn:local:.:60-c2iy93f"">Level2
                     Plantain PDS KO vector final assembly</a>)"
                     /Alternative_Match="100.00%; Adjusted-Interval-0:0/0;
                     OsU6-2 promoter (<a
                     href=""urn:local:.:52-c2iy3lf"">L1_target1 in pTwist Amp
                     High Copy - fake primer PDS target1</a>)"
                     /label="OsU6-2 promoter"
     CDS             1142..1465
                     /kind="reporter"
                     /note="alpha fragment of lacZ from pUC19 cloning vector.
                     Used for alpha complementation alongside omega fragment
                     (usually in host)"
                     /product="β-galactosidase"
                     /gene="lacZα"
                     /note="Derived using Geneious Prime 2020.1.2 'Annotate
                     from Database' based on nucleotide similarity"
                     /X-Transferred_From="lacZα"
                     /X-Transferred_Similarity="100.00%"
                     /X-Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:9e-bzo7tsh"">lacZα</a>"
                     /X-Alternative_Match="93.79%; Adjusted-Interval-0:54/-93;
                     <a href=""urn:local:.:8q-bzo7ts3"">lacZa</a>"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="L1_target2 in pTwist Amp High Copy"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; lacZα
                     (<a href=""urn:local:.:1f-c2incxl"">L1_target2 in pTwist
                     Amp High Copy</a>)"
                     /Alternative_Match="100.00%; Adjusted-Interval-0:0/0;
                     lacZα (<a href=""urn:local:.:1b-c2incxi"">L1_target1 in
                     pTwist Amp High Copy</a>)"
                     /modified_by="Florian"
                     /label="lacZα"
     misc_RNA        1562..1647
                     /created_by="Florian"
                     /label="improved sgRNA backbone (Dang et al., 2015)"
     terminator      1648..1656
                     /created_by="Florian"
                     /label="PolyT"
     Misc._feature   complement(1688..1815)
                     /vntifkey=21
                     /note="Unknown feature type:Misc._feature"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="pICSL11059 35Sp::HPTII:35St"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; RB short
                     (<a href=""urn:local:.:7jr-d1yz7zq"">pICSL11059
                     35Sp::HPTII:35St</a>)"
                     /Alternative_Match="100.00%; Adjusted-Interval-0:0/0; RB
                     short (<a href=""urn:local:.:w-c2ilwp9"">pFH52</a>)"
                     /Alternative_Match="100.00%; Adjusted-Interval-0:0/0; RB
                     short (<a href=""urn:local:.:f-c2iltpe"">pICSL11059</a>)"
                     /Alternative_Match="100.00%; Adjusted-Interval-0:0/0; RB
                     short (<a href=""urn:local:.:7jz-d1yz8pq"">pFH52
                     2x35Sp::SpCas9:NOSt</a>)"
                     /label="RB short"
     primer_bind     complement(1802..1827)
                     /%GC="53.8"
                     /Hairpin_Tm="54.9"
                     /Self_Dimer_Tm="21.4"
                     /Tm="66.5"
                     /annotation_group="1214696249109600de7b622b-9cb8-4bc7-a9b8
                     -d6f0d067b0de"
                     /created_by="primer3"
                     /Sequence="GAACCCTGTGGTTGGCATGCACATAC"
                     /Mismatches=0
                     /#_Local_Off-target_Sites=0
                     /Local_Off-target="pGMF2-M"
                     /label="GMF001"
     rep_origin      complement(2267..2881)
                     /vntifkey=21
                     /note="Derived using Geneious Prime 2020.2.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /X-Transferred_From="pFH37"
                     /X-Transferred_Similarity="99.84%"
                     /X-Primary_Match="99.84%; Adjusted-Interval-0:0/0; rep
                     (pMB1) (<a href=""urn:local:.:57l-bzp6ecj"">pFH37</a>)"
                     /X-Alternative_Match="99.84%; Adjusted-Interval-0:0/0; rep
                     (pMB1) (<a href=""urn:local:.:56e-bzp6ece"">pFH63</a>)"
                     /X-Alternative_Match="99.84%; Adjusted-Interval-0:0/0; rep
                     (pMB1) (<a href=""urn:local:.:57h-bzp6ecj"">pFH35</a>)"
                     /X-Alternative_Match="99.84%; Adjusted-Interval-0:0/0; rep
                     (pMB1) (<a href=""urn:local:.:56p-bzp6ecg"">pFH132</a>)"
                     /X-Alternative_Match="99.84%; Adjusted-Interval-0:0/0; rep
                     (pMB1) (<a href=""urn:local:.:567-bzp6ece"">pFH69</a>)"
                     /X-Alternative_Match="99.84%; Adjusted-Interval-0:0/0; rep
                     (pMB1) (<a href=""urn:local:.:56m-bzp6ecf"">pFH23 </a>)"
                     /X-Alternative_Match="99.84%; Adjusted-Interval-0:0/0; rep
                     (pMB1) (<a href=""urn:local:.:56i-bzp6ecf"">pFH53</a>)"
                     /X-Alternative_Match="99.84%; Adjusted-Interval-0:0/0; rep
                     (pMB1) (<a href=""urn:local:.:57o-bzp6ecj"">pFH38</a>)"
                     /X-Alternative_Match="99.84%; Adjusted-Interval-0:0/0; rep
                     (pMB1) (<a href=""urn:local:.:57k-bzp6ecj"">pFH34</a>)"
                     /X-Alternative_Match="99.84%; Adjusted-Interval-0:0/0; rep
                     (pMB1) (<a href=""urn:local:.:56d-bzp6ece"">pFH58</a>)"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="pUCIDT (Amp)"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; rep
                     (pMB1) (<a href=""urn:local:.:1s-ce2z606"">pUCIDT
                     (Amp)</a>)"
                     /Alternative_Match="100.00%; Adjusted-Interval-0:-11/15;
                     ori (<a href=""urn:local:.:ix-cgdmhmr"">pUCIDT-Amp
                     GoldenGate </a>)"
                     /label="rep (pMB1)"
     CDS             complement(3041..3901)
                     /note="Ampicillin"
                     /function="Drug Resistance"
                     /modified_by="Florian"
                     /label="Ampicillin"
     promoter        complement(3902..4006)
                     /created_by="hahnf"
                     /note="Derived using Geneious Prime 2020.2.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /X-Transferred_From="pFH114"
                     /X-Transferred_Similarity="99.05%"
                     /X-Primary_Match="99.05%; Adjusted-Interval-0:0/0; AmpR
                     promoter (<a href=""urn:local:.:56t-bzp6ecg"">pFH114</a>)"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="pUCIDT (Amp)"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; AmpR
                     promoter (<a href=""urn:local:.:1s-ce2z606"">pUCIDT
                     (Amp)</a>)"
                     /Alternative_Match="100.00%; Adjusted-Interval-0:0/0; AmpR
                     promoter (<a href=""urn:local:.:ix-cgdmhmr"">pUCIDT-Amp
                     GoldenGate </a>)"
                     /label="AmpR promoter"
ORIGIN      
        1 tcgcgcgttt cggtgcacga ggccctttca tctcgcgcgt ttcggtgatg acggtgaaaa
       61 cctctgacac atgcagctcc cctagacggt cacagcttgt ctgtaagcgg atgccgggag
      121 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
      181 tgcggcatca gagcagattg tactgagagt gcaccaaatg cggtgtgaaa taccgcacag
      241 atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg
      301 ggaagggcga tcggtgcggg cctcatcgct attacgccag ctggcgaaag ggggatgtgc
      361 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
      421 ggccagtgca acgcgatgac gatggatagc gattcatcga tgagctgacc cgatcgccgc
      481 cgccggaggg ttgcgtttga gacaggcgac agattctgac tgatgggctg cctgtatcga
      541 gtggtgattt tgtgccgagc tgccggtcgg ggagctgttg gctggctggt ggcaggatat
      601 attgtggtgt aaacaaattg acgcttagac aacttaataa cacattgcgg acgtttttaa
      661 tgtactgggg tggatgcagt gggccccact ctgtgaagac aattacgaat tcccatgggg
      721 agggatcatg aaccaacggc ctggctgtat ttggtggttg tgtagggaga tggggagaag
      781 aaaagcccga ttctcttcgc tgtgatgggc tggatgcatg cgggggagcg ggaggcccaa
      841 gtacgtgcac ggtgagcggc ccacagggcg agtgtgagcg cgagaggcgg gaggaacagt
      901 ttagtaccac attgcccagc taactcgaac gcgaccaact tataaacccg cgcgctgtcg
      961 cttgtgttgt gagaccgcag ctggcacgac aggtttgccg actggaaagc gggcagtgag
     1021 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     1081 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     1141 tatgaccatg attacgccaa gcttgcatgc ctgcaggtcg actctagagg atccccgggt
     1201 accgagctcg aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     1261 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     1321 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat
     1381 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
     1441 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     1501 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgacggtctc
     1561 agtttcagag ctatgctgga aacagcatag caagttgaaa taaggctagt ccgttatcaa
     1621 cttgaaaaag tggcaccgag tcggtgcttt ttttttcgct gggattgtct tctgcacgaa
     1681 gtggtttaaa ctatcagtgt ttgacaggat atattggcgg gtaaacctaa gagaaaagag
     1741 cgtttattag aataatcgga tatttaaaag ggcgtgaaaa ggtttatccg ttcgtccatt
     1801 tgtatgtgca tgccaaccac agggttcccc aatcagttct ggaccagcga gctgtgctgc
     1861 gactcgtggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     1921 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     1981 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     2041 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     2101 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     2161 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     2221 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     2281 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     2341 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     2401 gctctcctgt tccgaccctg tcgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     2461 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     2521 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     2581 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     2641 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     2701 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     2761 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     2821 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     2881 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     2941 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3001 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     3061 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     3121 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     3181 gagatccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     3241 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     3301 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     3361 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     3421 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     3481 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     3541 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     3601 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     3661 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     3721 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     3781 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     3841 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     3901 tactctacct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     3961 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     4021 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     4081 gtatcacgag gccctttcat c
//
