LOCUS       pJAT32{dU6-3-attR3-Amp-ccdB-attR4-MCS-attR5-Chlor-SacB-attR6-dU6-1}        8792 bp    DNA     circular UNA 12-JUN-2023
DEFINITION  Gibson/Homology Cloning ligation product.
ACCESSION   urn.local.sternd.1ts-gdrdirs
VERSION     urn.local.sternd.1ts-gdrdirs
KEYWORDS    .
SOURCE      
  ORGANISM  .
FEATURES             Location/Qualifiers
     CDS             411..1202
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKP
                     HSDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYP
                     AKRELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQ
                     DLFEALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPA
                     QYQPVILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
                     /standard_name="SmR"
     rep_origin      1448..2036
     regulatory      2239..2258
                     /note="Geneious type: promoter"
                     /note="SP6 RNA polymerase promoter"
                     /regulatory_class="promoter"
                     /note="Derived using Geneious Prime 2021.1.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /standard_name="SP6"
     regulatory      2334..2754
                     /note="Geneious type: promoter"
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
                     /standard_name="dU6-3 promoter"
     misc_feature    2761..2884
                     /note="Geneious type: attR3"
                     /standard_name="attR3"
     regulatory      2980..3084
                     /note="Geneious type: promoter"
                     /note="Promoter for ampicillin resistance gene
                     (b-lactamase)"
                     /regulatory_class="promoter"
                     /note="Derived using Geneious Prime 2021.1.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /standard_name="amp"
     gene            3085..3945
                     /standard_name="AmpR"
     gene            4255..4587
                     /standard_name="ccdB"
     misc_feature    complement(4625..4748)
                     /note="Geneious type: attR4"
                     /standard_name="attR4"
     misc_feature    4756..4759
                     /note="4 base BsaI overhang created during Golden Gate
                     assembly (BsaI site 320-325)"
                     /standard_name="BsaI overhang"
     misc_feature    4760..4901
                     /note="DNA sequence removed and replaced during Golden
                     Gate assembly with BsaI"
                     /standard_name="Fragment removed by BsaI"
     misc_feature    4761..4766
                     /standard_name="BsaI"
     misc_feature    4767..4770
                     /note="4 base BbsI overhang created during Golden Gate
                     assembly (BbsI site 343-348)"
                     /standard_name="BbsI overhang"
     misc_feature    4771..4890
                     /note="DNA sequence removed and replaced during Golden
                     Gate assembly with BbsI"
                     /standard_name="Fragment removed by BbsI"
     misc_feature    4773..4778
                     /standard_name="BbsI"
     misc_feature    complement(4883..4888)
                     /standard_name="BbsI"
     misc_feature    complement(4891..4894)
                     /note="4 base BbsI overhang created during Golden Gate
                     assembly (BbsI site 372-377)"
                     /standard_name="BbsI overhang"
     misc_feature    complement(4895..4900)
                     /standard_name="BsaI"
     misc_feature    complement(4902..4905)
                     /note="4 base BsaI overhang created during Golden Gate
                     assembly (BsaI site 395-400)"
                     /standard_name="BsaI overhang"
     misc_feature    4906..5029
                     /note="Geneious type: attR5"
                     /standard_name="attR5"
     gene            5136..5795
                     /gene="CAT/CamR"
                     /standard_name="CAT/CamR"
     regulatory      5804..6249
                     /note="Geneious type: promoter"
                     /note="Bacillus subtilis sacB (glycoside hydrolase family
                     68) promoter region"
                     /regulatory_class="promoter"
                     /note="Derived using Geneious Prime 2021.1.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /standard_name="sacB"
     gene            6250..7673
                     /note="counterselectable marker from Bacillus subtilis
                     that converts sucrose to levans"
                     /product="levansucrase"
                     /gene="sacB"
                     /note="Derived using Geneious Prime 2021.1.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /standard_name="sacB CS"
     misc_feature    complement(7710..7833)
                     /note="Geneious type: attR6"
                     /standard_name="attR6"
     regulatory      complement(7886..8689)
                     /note="Geneious type: promoter"
                     /standard_name="U6:1 promoter"
     regulatory      complement(8733..8750)
                     /regulatory_class="promoter"
                     /note="T7 promoter"
                     /standard_name="T7 promoter"
ORIGIN      
        1 gactttgctc atgttaccga tgctattcgg aagaacggca actaagctgc cgggtttgaa
       61 acacggatga tctcgcggag ggtagcatgt tgattgtaac gatgacagag cgttgctgcc
      121 tgtgatcaat tcgggcacga acccagtgga cataagcctg ttcggttcgt aagctgtaat
      181 gcaagtagcg tatgcgctca cgcaactggt ccagaacctt gaccgaacgc agcggtggta
      241 acggcgcagt ggcggttttc atggcttgtt atgactgttt ttttggggta cagtctatgc
      301 ctcgggcatc caagcagcaa gcgcgttacg ccgtgggtcg atgtttgatg ttatggagca
      361 gcaacgatgt tacgcagcag ggcagtcgcc ctaaaacaaa gttaaacatc atgggggaag
      421 cggtgatcgc cgaagtatcg actcaactat cagaggtagt tggcgtcatc gagcgccatc
      481 tcgaaccgac gttgctggcc gtacatttgt acggctccgc agtggatggc ggcctgaagc
      541 cacacagtga tattgatttg ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc
      601 gagctttgat caacgacctt ttggaaactt cggcttcccc tggagagagc gagattctcc
      661 gcgctgtaga agtcaccatt gttgtgcacg acgacatcat tccgtggcgt tatccagcta
      721 agcgcgaact gcaatttgga gaatggcagc gcaatgacat tcttgcaggt atcttcgagc
      781 cagccacgat cgacattgat ctggctatct tgctgacaaa agcaagagaa catagcgttg
      841 ccttggtagg tccagcggcg gaggaactct ttgatccggt tcctgaacag gatctatttg
      901 aggcgctaaa tgaaacctta acgctatgga actcgccgcc cgactgggct ggcgatgagc
      961 gaaatgtagt gcttacgttg tcccgcattt ggtacagcgc agtaaccggc aaaatcgcgc
     1021 cgaaggatgt cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca
     1081 tacttgaagc tagacaggct tatcttggac aagaagaaga tcgcttggcc tcgcgcgcag
     1141 atcagttgga agaatttgtc cactacgtga aaggcgagat caccaaggta gtcggcaaat
     1201 aatgtctagc tagaaattcg ttcaagccga cgccgcttcg cggcgcggct taactcaagc
     1261 gttagatgca ctaagcacat aattgctcac agccaaacta tcaggtcaag tctgctttta
     1321 ttatttttaa gcgtgcataa taagccctac acaaattggg agatatatca tgcatgacca
     1381 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     1441 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     1501 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     1561 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     1621 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     1681 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     1741 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     1801 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     1861 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     1921 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     1981 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     2041 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     2101 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     2161 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa atcgtcacct
     2221 gcaggaaggt ttaaacgcat ttaggtgaca ctatagaagt gtgtatcgct cgagggatcc
     2281 gaattcgaag tcttggtacg gaggggatcc gatcgacaag ccgaacatat gggttttttg
     2341 ctcacctgtg attgctccta ctcaaataca aaaacatcaa attttctgtc aataaagcat
     2401 atttatttat atttatttta caggaaagaa ttccttttaa agtgtatttt aacctataat
     2461 gaaaaacgat taaaaaaaat acataaaata attcgaaaat ttttgaatag cccaggttga
     2521 taaaaattca tttcatacgt tttataactt atgcccctaa gtattttttg accatagtgt
     2581 ttcaattcta cattaatttt acagagtaga atgaaacgcc acctactcag ccaagaggcg
     2641 aaaaggttag ctcgccaagc agagagggcg ccagtgctca ctacttttta taattctcaa
     2701 cttctttttc cagactcagt tcgtatatat agacctattt tcaatttaac gtcggggcct
     2761 caactttgta taataaagtt gaacgagaaa cgtaaaatga tataaatatc aatatattaa
     2821 attagatttt gcataaaaaa cagactacat aatactgtaa aacacaacat atccagtcac
     2881 tatgggccgc attaggcacc ccaggcttta cactttatgc ttccggctcg tataatgtgt
     2941 ggattttgag ttaggatccg tcgagatttt caggagctac gcggaacccc tatttgttta
     3001 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     3061 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     3121 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     3181 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     3241 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     3301 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     3361 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     3421 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     3481 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     3541 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     3601 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     3661 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     3721 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     3781 tctggagccg gtgagcgtgg gtcccgcggt atcattgcag cactggggcc agatggtaag
     3841 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     3901 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaatggat ccggcttact
     3961 aaaagccaga taacagtatg cgtatttgcg cgctgatttt tgcggtataa gaatatatac
     4021 tgatatgtat acccgaagta tgtcaaaaag aggtatgcta tgaagcagcg tattacagtg
     4081 acagttgaca gcgacagcta tcagttgctc aaggcatata tgatgtcaat atctccggtc
     4141 tggtaagcac aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc tggaaagcgg
     4201 aaaatcagga agggatggct gaggtcgccc ggtttattga aatgaacggc tcttttgctg
     4261 acgagaacag gggctggtga aatgcagttt aaggtttaca cctataaaag agagagccgt
     4321 tatcgtctgt ttgtggatgt acagagtgat attatcgata caccgggccg tcgcatggtt
     4381 attccacttg cgtctgcgcg cttgctctca gataaagtct cccgtgaact ttacccggtg
     4441 gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag tgtgccggtt
     4501 tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat caaaaacgcc
     4561 attaacctga tgttctgggg aatataatgt caggctccct tatacacagc cagtctgcac
     4621 gtcgcatagt gactggatat gttgtgtttt acagtattat gtagtctgtt ttttatgcaa
     4681 aatctaattt aatatattga tatttatatc attttacgtt tctcgttcaa cttttctata
     4741 caaagttgtc cagctggagc gagaccggag tcgtcttcgc tttccgaatt cgagctcggt
     4801 acctcgcgaa tgcatctaga tatcagatct ggatcccggg cccgtcgact gcagaggcct
     4861 gcatgcaagc ttgataactt gtgaagacga ccatggtctc accatcaact ttgtatacaa
     4921 aagttgaacg agaaacgtaa aatgatataa atatcaatat attaaattag attttgcata
     4981 aaaaacagac tacataatac tgtaaaacac aacatatcca gtcactatgg gccgcattag
     5041 gcaccccagg ctttacactt tatgcttccg gctcgtataa tgtgtggatt ttgagttagg
     5101 atccgtcgag attttcagga gctaaggaag ctaaaatgga gaaaaaaatc actggatata
     5161 ccaccgttga tatatcccaa tggcatcgta aagaacattt tgaggcattt cagtcagttg
     5221 ctcaatgtac ctataaccag accgttcagc tggatattac ggccttttta aagaccgtaa
     5281 agaaaaataa gcacaagttt tatccggcct ttattcacat tcttgcccgc ctgatgaatg
     5341 ctcatccgga attccgtatg gcaatgaaag acggtgagct ggtgatatgg gatagtgttc
     5401 acccttgtta caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat
     5461 accacgacga tttccggcag tttctacaca tatattcgca agatgtggcg tgttacggtg
     5521 aaaacctggc ctatttccct aaagggttta ttgagaatat gtttttcgtg tcagccaatc
     5581 cctgggtgag tttcaccagt tttgatttaa acgtggccaa tatggacaac ttcttcgccc
     5641 ccgttttcac catgggcaaa tattatacgc aaggcgacaa ggtgctgatg ccgctggcga
     5701 ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt aatgaattac
     5761 aacagtactg cgatgagtgg cagggcgggg cgtaaacgcg tggcacatat acctgccgtt
     5821 cactattatt tagtgaaatg agatattatg atattttctg aattgtgatt aaaaaggcaa
     5881 ctttatgccc atgcaacaga aactataaaa aatacagaga atgaaaagaa acagatagat
     5941 tttttagttc tttaggcccg tagtctgcaa atccttttat gattttctat caaacaaaag
     6001 aggaaaatag accagttgca atccaaacga gagtctaata gaatgaggtc gaaaagtaaa
     6061 tcgcgcgggt ttgttactga taaagcaggc aagacctaaa atgtgtaaag ggcaaagtgt
     6121 atactttggc gtcacccctt acatatttta ggtctttttt tattgtgcgt aactaacttg
     6181 ccatcttcaa acaggagggc tggaagaagc agaccgctaa cacagtacat aaaaaaggag
     6241 acatgaacga tgaacatcaa aaagtttgca aaacaagcaa cagtattaac ctttactacc
     6301 gcactgctgg caggaggcgc aactcaagcg tttgcgaaag aaacgaacca aaagccatat
     6361 aaggaaacat acggcatttc ccatattaca cgccatgata tgctgcaaat ccctgaacag
     6421 caaaaaaatg aaaaatatca agttcctgaa ttcgattcgt ccacaattaa aaatatctct
     6481 tctgcaaaag gcctggacgt ttgggacagc tggccattac aaaacgctga cggcactgtc
     6541 gcaaactatc acggctacca catcgtcttt gcattagccg gagatcctaa aaatgcggat
     6601 gacacatcga tttacatgtt ctatcaaaaa gtcggcgaaa cttctattga cagctggaaa
     6661 aacgctggcc gcgtctttaa agacagcgac aaattcgatg caaatgattc tatcctaaaa
     6721 gaccaaacac aagaatggtc aggttcagcc acatttacat ctgacggaaa aatccgttta
     6781 ttctacactg atttctccgg taaacattac ggcaaacaaa cactgacaac tgcacaagtt
     6841 aacgtatcag catcagacag ctctttgaac atcaacggtg tagaggatta taaatcaatc
     6901 tttgacggtg acggaaaaac gtatcaaaat gtacagcagt tcatcgatga aggcaactac
     6961 agctcaggcg acaaccatac gctgagagat cctcactacg tagaagataa aggccacaaa
     7021 tacttagtat ttgaagcaaa cactggaact gaagatggct accaaggcga agaatcttta
     7081 tttaacaaag catactatgg caaaagcaca tcattcttcc gtcaagaaag tcaaaaactt
     7141 ctgcaaagcg ataaaaaacg cacggctgag ttagcaaacg gcgctctcgg tatgattgag
     7201 ctaaacgatg attacacact gaaaaaagtg atgaaaccgc tgattgcatc taacacagta
     7261 acagatgaaa ttgaacgcgc gaacgtcttt aaaatgaacg gcaaatggta cctgttcact
     7321 gactcccgcg gatcaaaaat gacgattgac ggcattacgt ctaacgatat ttacatgctt
     7381 ggttatgttt ctaattcttt aactggccca tacaagccgc tgaacaaaac tggccttgtg
     7441 ttaaaaatgg atcttgatcc taacgatgta acctttactt actcacactt cgctgtacct
     7501 caagcgaaag gaaacaatgt cgtgattaca agctatatga caaacagagg attctacgca
     7561 gacaaacaat caacgtttgc gccaagcttc ctgctgaaca tcaaaggcaa gaaaacatct
     7621 gttgtcaaag acagcatcct tgaacaagga caattaacag ttaacaaata aatgtcaggc
     7681 tcccttatac acagccagtc tgcacgtcgc atagtgactg gatatgttgt gttttacagt
     7741 attatgtagt ctgtttttta tgcaaaatct aatttaatat attgatattt atatcatttt
     7801 acgtttctcg ttcaactttt taatacaaag ttgcctccgg aacataatgg tgcatctaga
     7861 tgcccattcc tgtagacttc ttaatcgaag ttcacccgga tatctttcct atatatactg
     7921 tactcttgca gaatatgtac ttgggaatca taaggatgcg aaggacgtac gcgctgtgtc
     7981 tgtaactaag gggttgcttt cgggtaccta ccatgccaat gatgggacta ccgccggata
     8041 ggaatccctt ttgggtgcac accgtcacct ccagttccgt ctgcctgagt tcggcaatgg
     8101 aaatcaggta ctcaaaggat gcatcgtaga cggggttgca gttgtccttg atcacgctcg
     8161 tcttgcgttt cgactccttg gtgcgtccag gcaacagata cagcttaaca tacggatcgg
     8221 ggatattgct gggatcgcga agtggtatct tctggatttt gtgtatggtc acgtctagtt
     8281 tttgacgctg ggcgctgtag cggatcgaca actgcattcg tccaaggcca tgttcgccgg
     8341 gtgaggaggt ggagtctgga aagcggtggg tcagcacaga ggtggccagc tcgctgatgg
     8401 gtttttcgct ggaggtggag gccgacatgg ccggggagat tttcgtagcc gccacaggat
     8461 cttcttcact gatgggcgat tcggcggaca aacgcttgct ggatgcctga gattcctaaa
     8521 agcaatggca aattagattc aattaaaaca gaaattgttt atataacaga gtaaaagcaa
     8581 aaaaaaagga atcacagatt ctccccgacg gggaattgaa ccccgttctc ccgcgtgaca
     8641 ggcggggata ctaaccacta tactatcgag gacgttgaaa atgaattcaa ttaagacgtc
     8701 agaattctcg aggcggccgc atgtgagtct ccctatagtg agtcgtatta atttcgcggg
     8761 cgcgtcagcg ggagagccgt tgtaaggcgg ca
//
