LOCUS       pJAT27{dU6-3-attR3-Amp-ccdB-attR4-MHC-GFPc-attR5-Chlor-SacB-attR6}       10147 bp    DNA     circular UNA 12-JUN-2023
DEFINITION  Golden Gate/Type IIS Cloning ligation product.
ACCESSION   urn.local.sternd.21t-gdru7ip
VERSION     urn.local.sternd.21t-gdru7ip
KEYWORDS    .
SOURCE      
  ORGANISM  .
FEATURES             Location/Qualifiers
     misc_feature    1..4770
                     /note="Geneious type: Concatenated sequence"
                     /standard_name="P859
                     p{dU6-3-GW1-Amp-ccdB-MCS-GW2-Chlor-SacB} extraction
                     (concatenated sequence 1)"
     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
     misc_feature    1820..1837
                     /note="Geneious type: primer_bind_reverse"
                     /note="Derived using Geneious Prime 2021.1.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /standard_name="ori-252 R"
     primer_bind     1937..1956
                     /note="pBR322 origin, forward primer"
                     /standard_name="pBR322ori-F"
     primer_bind     2026..2045
                     /note="Derived using Geneious Prime 2021.1.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /standard_name="T7Cas9F"
     primer_bind     complement(2052..2071)
                     /note="Derived using Geneious Prime 2021.1.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /standard_name="amp-ori-7571F"
     misc_feature    2180..2209
                     /note="Geneious type: ligation"
                     /standard_name="Ligation"
     primer_bind     2190..2207
                     /note="L4440 vector, forward primer"
                     /standard_name="L4440"
     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"
     misc_feature    2300..2303
                     /note="Geneious type: ligation"
                     /standard_name="Ligation"
     misc_feature    2304..2333
                     /note="Geneious type: Editing History Insertion"
                     /standard_name
     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    2755..2787
                     /note="Geneious type: Editing History Insertion"
                     /standard_name
     misc_feature    2756
                     /note="Geneious type: Junction"
                     /standard_name="junction 1"
     misc_feature    2757
                     /note="Geneious type: Junction"
                     /standard_name="junction 1"
     misc_feature    2758..2787
                     /note="Geneious type: ligation"
                     /standard_name="Ligation"
     misc_feature    2761..2884
                     /note="Geneious type: attR3"
                     /standard_name="attR3"
     regulatory      2907..2937
                     /note="Geneious type: promoter"
                     /note="Lac promoter of Escherichia coli containing 2 base
                     pair mutations"
                     /regulatory_class="promoter"
                     /note="Derived using Geneious Prime 2021.2.2 'Annotate
                     from Database' based on nucleotide similarity"
                     /standard_name="lacUV5"
     misc_feature    complement(2918..2929)
                     /note="C-tag used for protein purification"
                     /note="Derived using Geneious Prime 2021.2.2 'Annotate
                     from Database' based on protein similarity"
                     /standard_name="C"
     misc_feature    2976..2979
                     /note="Geneious type: ligation"
                     /standard_name="Ligation"
     misc_feature    2979
                     /note="Geneious type: Junction"
                     /standard_name="junction 2"
     misc_feature    2979
                     /note="Geneious type: Junction"
                     /standard_name="junction 1"
     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"
     primer_bind     complement(3125..3146)
                     /note="Derived using Geneious Prime 2021.1.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /standard_name="AMP_5'_F"
     misc_feature    3331..3350
                     /note="Geneious type: primer_bind_reverse"
                     /note="Derived using Geneious Prime 2021.1.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /standard_name="Amp+_R"
     misc_feature    3804
                     /note="Geneious type: Editing History Replacement"
                     /standard_name="T"
     primer_bind     complement(3817..3836)
                     /note="Derived using Geneious Prime 2021.1.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /standard_name="Amp+_F"
     misc_feature    3909..3932
                     /note="Geneious type: primer_bind_reverse"
                     /note="Derived using Geneious Prime 2021.1.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /standard_name="Amp_3'_R"
     primer_bind     complement(3922..3945)
                     /standard_name="966 R"
     misc_feature    3942..3945
                     /note="Geneious type: ligation"
                     /standard_name="Ligation"
     misc_feature    3946..4585
                     /note="Geneious type: Editing History Replacement"
                     /standard_name="AGCAGCGTATTACAGTGACAGTTGACAGCGACAGCTATCAGT
                     TGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGAAT
                     GAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGATGGCTG
                     AGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAGGGGCTGGTG
                     AAATGCAATTTAAGGTTTATACTTACAAGCGCGAGTCCCGCTATCGCCTTTTCGTGGA
                     CGTCCAGTCAGACATTATCGACACGCCTGGTCGCCGTATGGTGATTCCATTAGCCAGC
                     GCACGCCTGTTAAGTGATAAGGTGTCGCGCGAGCTTTACCCAGTCGTCCATATTGGTG
                     ACGAGAGTTGGCGCATGATGACTACAGACATGGCTAGTGTACCTGTGTCGGTGATCGG
                     GGAAGAGGTCGCGGATTTAAGCCACCGTGAGAATGATATTAAGAATGCAATCAATCTC
                     ATGTTTTGGGGTATTTA"
     primer_bind     3946..3967
                     /standard_name="1 F"
     gene            4255..4587
                     /standard_name="ccdB"
     primer_bind     4446..4465
                     /standard_name="4446F"
     misc_feature    4585
                     /note="Geneious type: Junction"
                     /standard_name="junction 1"
     misc_feature    complement(4625..4748)
                     /note="Geneious type: attR4"
                     /standard_name="attR4"
     primer_bind     4741..4760
                     /standard_name="4741F"
     misc_feature    4748
                     /note="Geneious type: Junction"
                     /standard_name="junction 2"
     misc_feature    4750
                     /note="Geneious type: Junction"
                     /standard_name="junction 2"
     misc_feature    join(4752..>4770,<7052..7070)
                     /note="Geneious type: Concatenated sequence"
                     /standard_name="pGGAselect-Spect extraction (concatenated
                     sequence 2)"
     misc_feature    4752..4755
                     /note="Geneious type: ligation"
                     /standard_name="Ligation"
     misc_feature    4756..4759
                     /note="4 base BsaI overhang created during Golden Gate
                     assembly (BsaI site 320-325)"
                     /standard_name="BsaI overhang"
     misc_feature    join(<4756..>4770,<7052..>7066)
                     /note="Geneious type: Concatenated sequence"
                     /standard_name="pGGAselect PCR Product (concatenated
                     sequence 2)"
     misc_feature    join(4760..>4770,<7052..7062)
                     /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    complement(4767..5636)
                     /note="Geneious type: Concatenated sequence"
                     /standard_name="P514 pUC57{Mhc-678::dsRed} extraction
                     (concatenated sequence 2) (reversed)"
     misc_feature    4767..4770
                     /note="Geneious type: ligation"
                     /standard_name="Ligation"
     regulatory      4771..5448
                     /note="Geneious type: Enhancer"
                     /standard_name="MHC-678 enhancer"
     misc_feature    <4771..5473
                     /note="Geneious type: Concatenated sequence"
                     /standard_name="Mhc_F4-678 (concatenated sequence 2)"
     primer_bind     4771..4792
                     /standard_name="1 F"
     misc_feature    <4771..>5448
                     /note="Geneious type: golden_path_region"
                     /standard_name="2L"
     misc_feature    <4771..>5448
                     /note="Geneious type: exon_junction"
                     /standard_name="Dmel:r6:2L:16766756:16768584:"
     mRNA            <4771..>5448
                     /standard_name="Mhc-RR"
     misc_feature    <4771..>5448
                     /note="Geneious type: BAC_cloned_genomic_insert"
                     /standard_name="CH321-45C21"
     gene            <4771..>5448
                     /standard_name="Mhc"
     misc_feature    <4771..>5448
                     /note="Geneious type: origin_of_replication"
                     /standard_name="eORI_mE_98"
     misc_feature    <4771..>5448
                     /note="Geneious type: BAC_cloned_genomic_insert"
                     /standard_name="CH322-118J19"
     misc_feature    <4771..>5448
                     /note="Geneious type: BAC_cloned_genomic_insert"
                     /standard_name="CH322-104N7"
     misc_feature    <4771..>5448
                     /note="Geneious type: BAC_cloned_genomic_insert"
                     /standard_name="CH321-60K2"
     misc_feature    <4771..>5448
                     /note="Geneious type: BAC_cloned_genomic_insert"
                     /standard_name="CH322-167G4"
     misc_feature    <4771..>5448
                     /note="Geneious type: exon_junction"
                     /standard_name="Dmel:r6:2L:16767130:16768584:"
     misc_feature    <4771..>5448
                     /note="Geneious type: BAC_cloned_genomic_insert"
                     /standard_name="CH322-166E7"
     misc_feature    <4771..>5448
                     /note="Geneious type: BAC_cloned_genomic_insert"
                     /standard_name="CH321-49C23"
     misc_feature    <4771..>5448
                     /note="Geneious type: chromosome_band"
                     /standard_name="band-36B"
     misc_feature    <4771..>5448
                     /note="Geneious type: syntenic_region"
                     /standard_name="syntenic_region"
     misc_feature    <4771..>5448
                     /note="Geneious type: orthologous_to"
                     /standard_name="Mhc"
     misc_feature    <4771..>5448
                     /note="Geneious type: BAC_cloned_genomic_insert"
                     /standard_name="CH321-68G12"
     misc_feature    <4771..>5448
                     /note="Geneious type: BAC_cloned_genomic_insert"
                     /standard_name="CH322-151N23"
     misc_feature    <4771..>5448
                     /note="Geneious type: chromosome_band"
                     /standard_name="band-36"
     misc_feature    <4771..>5448
                     /note="Geneious type: BAC_cloned_genomic_insert"
                     /standard_name="CH322-150H3"
     misc_feature    <4771..>5448
                     /note="Geneious type: BAC_cloned_genomic_insert"
                     /standard_name="BACR26O03"
     misc_feature    <4771..>5448
                     /note="Geneious type: chromosome_band"
                     /standard_name="band-36B1"
     misc_feature    <4771..>5448
                     /note="Geneious type: BAC_cloned_genomic_insert"
                     /standard_name="CH322-12J14"
     misc_feature    <4771..>5448
                     /note="Geneious type: regulatory_region"
                     /standard_name="Mhc_mhc2"
     misc_feature    <4771..>5448
                     /note="Geneious type: regulatory_region"
                     /standard_name="Mhc_mhc1.5"
     misc_feature    <4771..4906
                     /note="Geneious type: regulatory_region"
                     /standard_name="Mhc_F4-728"
     misc_feature    <4771..>5448
                     /note="Geneious type: BAC_cloned_genomic_insert"
                     /standard_name="CH321-11A2"
     misc_feature    <4771..5335
                     /note="Geneious type: regulatory_region"
                     /standard_name="MHC_mhc1.2"
     misc_feature    4771..5448
                     /note="Geneious type: regulatory_region"
                     /standard_name="Mhc_F4-678"
     misc_feature    <4771..>5448
                     /note="Geneious type: BAC_cloned_genomic_insert"
                     /standard_name="CH321-62P22"
     misc_feature    <4771..>5448
                     /note="Geneious type: exon_junction"
                     /standard_name="Dmel:r6:2L:16766776:16768584:"
     misc_feature    <4771..>5448
                     /note="Geneious type: exon_junction"
                     /standard_name="Dmel:r6:2L:16766821:16768584:"
     mRNA            <4771..>5448
                     /standard_name="Mhc-RN"
     mRNA            <4771..>5448
                     /standard_name="Mhc-RO"
     mRNA            <4771..>5448
                     /standard_name="Mhc-RP"
     misc_feature    <4771..>5448
                     /note="Geneious type: BAC_cloned_genomic_insert"
                     /standard_name="CH321-50B3"
     mRNA            <4771..>5448
                     /standard_name="Mhc-RE"
     mRNA            <4771..>5448
                     /standard_name="Mhc-RC"
     mRNA            <4771..>5448
                     /standard_name="Mhc-RB"
     mRNA            <4771..>5448
                     /standard_name="Mhc-RA"
     mRNA            <4771..>5448
                     /standard_name="Mhc-RG"
     mRNA            <4771..>5448
                     /standard_name="Mhc-RM"
     mRNA            <4771..>5448
                     /standard_name="Mhc-RK"
     mRNA            <4771..>5448
                     /standard_name="Mhc-RL"
     mRNA            <4771..>5448
                     /standard_name="Mhc-RI"
     mRNA            <4771..>5448
                     /standard_name="Mhc-RH"
     mRNA            <4771..>5448
                     /standard_name="Mhc-RF"
     mRNA            <4771..>5448
                     /standard_name="Mhc-RD"
     intron          <4771..>5448
                     /standard_name="intron"
     misc_feature    4811..5011
                     /note="Geneious type: TF_binding_site"
                     /standard_name="ChIP-chip_Mef2_E8-10h_000105"
     misc_feature    4854..5054
                     /note="Geneious type: TF_binding_site"
                     /standard_name="ChIP-chip_Mef2_E10-12h_000119"
     misc_feature    4918..5181
                     /note="Geneious type: transcription_start_site"
                     /standard_name="TSS_RAMPAGE_001727"
     mRNA            5017..>5448
                     /standard_name="Mhc-RQ"
     mRNA            5017..>5448
                     /standard_name="Mhc-RS"
     mRNA            5017..>5448
                     /standard_name="Mhc-RU"
     mRNA            5017..>5448
                     /standard_name="Mhc-RV"
     exon            5017..5199
                     /standard_name="exon"
     5'UTR           5017..5199
                     /standard_name="5'UTR"
     exon            5017..5194
                     /standard_name="exon"
     5'UTR           5017..5194
                     /standard_name="5'UTR"
     exon            5017..5181
                     /standard_name="exon"
     5'UTR           5017..5181
                     /standard_name="5'UTR"
     misc_feature    5181..>5448
                     /note="Geneious type: exon_junction"
                     /standard_name="Dmel:r6:2L:16767941:16768584:"
     intron          5182..>5448
                     /standard_name="intron"
     misc_feature    5194..>5448
                     /note="Geneious type: exon_junction"
                     /standard_name="Dmel:r6:2L:16767954:16768584:"
     intron          5195..>5448
                     /standard_name="intron"
     misc_feature    5199..>5448
                     /note="Geneious type: exon_junction"
                     /standard_name="Dmel:r6:2L:16767959:16768584:"
     intron          5200..>5448
                     /standard_name="intron"
     misc_feature    5334..>5448
                     /note="Geneious type: regulatory_region"
                     /standard_name="Mhc_F4-453"
     misc_feature    5370..>5448
                     /note="Geneious type: TF_binding_site"
                     /standard_name="ChIP-chip_bin_E10-12h_000074"
     misc_feature    5429..>5448
                     /note="Geneious type: BAC_cloned_genomic_insert"
                     /standard_name="CH322-18B14"
     misc_feature    5449..>5636
                     /note="Geneious type: Concatenated sequence"
                     /standard_name="Digestion of pUC57{3P3::DsRed} - Fragment
                     2 (concatenated sequence 1)"
     misc_feature    5449..5473
                     /note="Geneious type: ligation"
                     /standard_name="Ligation"
     regulatory      5459..5636
                     /note="Geneious type: Promoter"
                     /standard_name="HS prom"
     primer_bind     complement(5620..5636)
                     /standard_name="866 R"
     misc_feature    5632
                     /note="Geneious type: Junction"
                     /standard_name="junction 1"
     misc_feature    5633..7055
                     /note="Geneious type: Concatenated sequence"
                     /standard_name="MBP-GFPc-SV40 extraction (concatenated
                     sequence 3)"
     misc_feature    5633..5636
                     /note="Geneious type: ligation"
                     /standard_name="Ligation"
     misc_feature    5636
                     /note="Geneious type: Junction"
                     /standard_name="junction 1"
     gene            5637..6803
                     /standard_name="MBP-GFPc"
     regulatory      6807..7051
                     /note="Geneious type: Terminator"
                     /standard_name="SV40_PA term"
     misc_feature    6807..7051
                     /note="Geneious type: Concatenated sequence"
                     /standard_name="SV40_PA term (concatenated sequence 3)"
     primer_bind     complement(7031..7051)
                     /standard_name="1,415 R"
     misc_feature    complement(7052..7055)
                     /note="4 base BbsI overhang created during Golden Gate
                     assembly (BbsI site 372-377)"
                     /standard_name="BbsI overhang"
     misc_feature    7052..10147
                     /note="Geneious type: Concatenated sequence"
                     /standard_name="P859
                     p{dU6-3-GW1-Amp-ccdB-MCS-GW2-Chlor-SacB} extraction
                     (concatenated sequence 4)"
     misc_feature    7052..7055
                     /note="Geneious type: ligation"
                     /standard_name="Ligation"
     misc_feature    7052
                     /note="Geneious type: Junction"
                     /standard_name="junction 1"
     misc_feature    7055^7056
                     /note="Geneious type: Editing History Deletion"
                     /standard_name="CGTCTCACCAT"
     misc_feature    complement(7056..7061)
                     /standard_name="BsaI"
     misc_feature    7056
                     /note="Geneious type: Junction"
                     /standard_name="junction 1"
     misc_feature    complement(7063..7066)
                     /note="4 base BsaI overhang created during Golden Gate
                     assembly (BsaI site 395-400)"
                     /standard_name="BsaI overhang"
     misc_feature    7063
                     /note="Geneious type: Junction"
                     /standard_name="junction 1"
     misc_feature    7064
                     /note="Geneious type: Junction"
                     /standard_name="junction 1"
     misc_feature    7067..7190
                     /note="Geneious type: attR5"
                     /standard_name="attR5"
     misc_feature    7067..4755
                     /note="Geneious type: Concatenated sequence"
                     /standard_name="P767
                     p{dU6-3-GW1-Amp-ccdB-Mhc-GFP-FRT-P3-DsRed-GW2-Chlor-SacB}
                     (concatenated sequence 1)"
     misc_feature    7067..7070
                     /note="Geneious type: ligation"
                     /standard_name="Ligation"
     misc_feature    7286
                     /note="Geneious type: Junction"
                     /standard_name="junction 2"
     misc_feature    7286
                     /note="Geneious type: Junction"
                     /standard_name="junction 1"
     gene            7297..7956
                     /gene="CAT/CamR"
                     /standard_name="CAT/CamR"
     misc_feature    7302..7954
                     /note="Geneious type: Editing History Replacement"
                     /standard_name="AAAAAAGATTACGGGTTATACTACCGTCGACATCAGCCAGTG
                     GCACCGTAAAGAACATTTCGAAGCGTTCCAGAGCGTCGCGCAGTGCACCTACAATCAG
                     ACCGTTCAGCTGGATATCACTGCTTTCCTGAAGACTGTTAAGAAAAACAAACACAAAT
                     TTTATCCGGCTTTTATCCACATTCTCGCTCGCCTGATGAACGCACACCCGGAATTCCG
                     TATGGCCATGAAAGATGGCGAGCTGGTAATTTGGGATTCCGTTCATCCGTGTTATACC
                     GTCTTTCACGAACAGACCGAGACGTTCTCTTCCCTGTGGTCTG"
     primer_bind     complement(7395..7414)
                     /note="Derived using Geneious Prime 2021.2.2 'Annotate
                     from Database' based on nucleotide similarity"
                     /standard_name="10,588 R"
     misc_feature    7959..9831
                     /note="Geneious type: Editing History Replacement"
                     /standard_name="CGAGACGTTCTCTTCCCTGTGGTCTGAATATCACGATGACTT
                     CCGTCAATTCCTGCACATTTATTCTCAGGACGTTGCGTGCTATGGTGAGAATCTCGCT
                     TATTTTCCGAAGGGTTTCATTGAAAACATGTTCTTTGTTAGCGCCAACCCATGGGTGA
                     GCTTCACTTCTTTCGACCTGAACGTGGCGAACATGGACAACTTTTTCGCGCCGGTTTT
                     CACGATGGGCAAGTATTACACCCAGGGTGACAAGGTACTGATGCCGCTGGCAATCCAG
                     GTGCATCACGCGGTATGTGACGGCTTCCATGTCGGTCGCATGCTGAACGAGCTGCAGC
                     AATACTGTGATGAATGGCAGGGCGGTGCGTAAACGCGTGGATCCGGCTTACTAAAAGC
                     CAGATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGAT
                     ATGTATACCCGAAGTATGTCAAAAAGAGGTATGCTATGAAGCAGCGTATTACAGTGAC
                     AGTTGACAGCGACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTC
                     TGGTAAGCACAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGC
                     GGAAAATCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTT
                     GCTGACGAGAACAGGGGCTGGTGAAATGCAATTTAAAGTTTATACCTACAAGCGCGAA
                     TCTCGCTACCGTCTGTTTGTAGACGTACAGTCTGATATCATTGATACGCCGGGCCGCC
                     GTATGGTTATTCCGCTGGCATCTGCACGTCTCCTGTCTGATAAAGTTAGCCGTGAACT
                     GTATCCGGTTGTACACATTGGCGACGAGTCTTGGCGCATGATGACGACTGACATGGCT
                     TCTGTCCCGGTTAGCGTCATTGGCGAGGAAGTCGCCGATCTGTCTCACCGTGAAAATG
                     ACATCAAAAATGCAATTAACCTGATGTTCTGGGGCATTTGATCCGGCTTACTAAAAGC
                     CAGATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGAT
                     ATGTATACCCGAAGTATGTCAAAAAGAGGTATGCTATGAAGCAGCGTATTACAGTGAC
                     AGTTGACAGCGACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTC
                     TGGTAAGCACAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGC
                     GGAAAATCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTT
                     GCTGACGAGAACAGGGGCTGGTGAAATGCAATTTAAAGTTTATACCTACAAGCGCGAA
                     TCTCGCTACCGTCTGTTTGTAGACGTACAGTCTGATATCATTGATACGCCGGGCCGCC
                     GTATGGTTATTCCGCTGGCATCTGCACGTCTCCTGTCTGATAAAGTTAGCCGTGAACT
                     GTATCCGGTTGTACACATTGGCGACGAGTCTTGGCGCATGATGACGACTGACATGGCT
                     TCTGTCCCGGTTAGCGTCATTGGCGAGGAAGTCGCCGATCTGTCTCACCGTGAAAATG
                     ACATCAAAAATGCAATTAACCTGATGTTCTGGGGCATTTG"
     misc_feature    7961..7964
                     /note="Geneious type: ligation"
                     /standard_name="Ligation"
     regulatory      7965..8410
                     /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            8411..9834
                     /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    8802..8805
                     /note="Geneious type: ligation"
                     /standard_name="Ligation"
     misc_feature    9832
                     /note="Geneious type: Junction"
                     /standard_name="junction 1"
     misc_feature    complement(9871..9994)
                     /note="Geneious type: attR6"
                     /standard_name="attR6"
     misc_feature    9995..10024
                     /note="Geneious type: Editing History Insertion"
                     /standard_name
     misc_feature    9995
                     /note="Geneious type: Junction"
                     /standard_name="junction 2"
     misc_feature    9997
                     /note="Geneious type: Junction"
                     /standard_name="junction 2"
     misc_feature    10025..10028
                     /note="Geneious type: ligation"
                     /standard_name="Ligation"
     regulatory      complement(10087..10105)
                     /note="Geneious type: Promoter"
                     /note="Derived using Geneious Prime 2021.2.2 'Annotate
                     from Database' based on nucleotide similarity"
                     /standard_name="T7 prom"
     regulatory      complement(10088..10105)
                     /regulatory_class="promoter"
                     /note="T7 promoter"
                     /standard_name="T7 promoter"
     misc_feature    10118..10147
                     /note="Geneious type: ligation"
                     /standard_name="Ligation"
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 atactcgaat tcgttgtcgg ctcatataca
     4801 tgggcgaaat aatttcgaat atgttttaaa aataaccaaa gacattagaa agagatcgcc
     4861 aatacttata cattatgtct atgtgtgcca tgtggtagca tgagccaaaa agcttctcga
     4921 aattacgaat tacatataga cgaatatgta tgtgacttta gtttcgaaat aatttcgaga
     4981 attttaaaaa taacgcattc gttaaaagtt cgcgtcaatt cgaatcgaat tttcgattgt
     5041 cgatttagtg tggattgtcg aaaatcgttc cgccttcgaa gtttactgaa aggaatcatt
     5101 gcgatctcgt gaattgcttg tatgagaaca cgccaccata tcgagatacc tgttcaaaat
     5161 atcgaaatcg cgttccaaac ggtagtagtt accagttgag tgagttgtgg tgacagttta
     5221 acctctctac atttgtaata ataagcattg ttttggcgta tcaatagtct gtagattttt
     5281 tcacaaagaa acctggagaa atgtacacaa atatctaaat gaaaattggc gttacgtaac
     5341 aaataccaac gccccgaaaa tccgtatata tgcaaagcat tctgatatat atatatataa
     5401 gtggtataca tatacatatt cgtatcggat ccgtgatcta tatttaggaa ccctcgaccg
     5461 ccggagtata aatagaggcg cttcgtctac ggagcgacaa ttcaattcaa acaagcaaag
     5521 tgaacacgtc gctaagcgaa agctaagcaa ataaacaagc gcagctgaac aagctaaaca
     5581 atcggggtac cgctagagtc gacggtaccg cgggcccggg atccaccggt cgccacatga
     5641 agatcgagga gggcaagttg gtcatctgga ttaatggaga taagggctat aatggactgg
     5701 cagaggttgg aaaaaaattt gagaaggata caggcattaa ggtcacagtt gagcatccag
     5761 ataagttgga ggagaagttc ccgcaggtgg ctgcgacggg tgacggaccc gacatcatat
     5821 tctgggctca tgaccgtttc ggaggatacg cccagtccgg cttgttggcg gaaattaccc
     5881 ctgataaagc ttttcaggac aaactctacc ccttcacctg ggatgctgtc cgatacaacg
     5941 gcaagctcat tgcatacccc attgcagttg aagcactgag tctgatttac aataaagacc
     6001 tgttgccgaa tcctccgaag acctgggagg agattcccgc tctggacaag gagctgaagg
     6061 ctaagggcaa gtccgccctg atgttcaacc tgcaagagcc gtactttact tggcccctga
     6121 tcgcagccga cggcggatac gccttcaaat acgagaacgg aaagtacgat ataaaggatg
     6181 tcggagtcga caacgccggc gctaaagcag gcctgacttt cctggtggac ctcataaaga
     6241 acaagcatat gaatgccgac accgattaca gtatagcgga agccgcgttc aacaagggcg
     6301 agactgccat gacaataaac ggaccctggg catggtccaa cattgacacg agcaaggtga
     6361 actacggtgt aacggtgttg cctacgttca agggacagcc gagtaaacca ttcgttggcg
     6421 ttttgtcggc tggaattaat gctgctagtc ccaataagga gttggctaag gagttcttgg
     6481 agaactactt gctgacagac gagggcttgg aggcagtaaa caaggataag cccctgggtg
     6541 cggttgccct caagtcctat gaagaagagc tcgtaaaaga cccgcgtatt gctgcgacca
     6601 tggagaatgc ccagaagggt gagattatgc ccaatatacc ccagatgtcc gcgttttggt
     6661 atgcagttcg caccgcggtt ataaacgcag cctcgggacg gcagaccgtg gatgaggact
     6721 tgaaagacgc ccagacacgc ataaccaaac agtttcgcga ccatatggtt ctgcatgaat
     6781 atgtcaacgc agcaggcatt acttaaagat cataatcagc cataccacat ttgtagaggt
     6841 tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc
     6901 aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     6961 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     7021 catcaatgta tcttaaagct tatcgatacg cccatggtct caccatcaac tttgtataca
     7081 aaagttgaac gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat
     7141 aaaaaacaga ctacataata ctgtaaaaca caacatatcc agtcactatg ggccgcatta
     7201 ggcaccccag gctttacact ttatgcttcc ggctcgtata atgtgtggat tttgagttag
     7261 gatccgtcga gattttcagg agctaaggaa gctaaaatgg agaaaaaaat cactggatat
     7321 accaccgttg atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt
     7381 gctcaatgta cctataacca gaccgttcag ctggatatta cggccttttt aaagaccgta
     7441 aagaaaaata agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat
     7501 gctcatccgg aattccgtat ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt
     7561 cacccttgtt acaccgtttt ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa
     7621 taccacgacg atttccggca gtttctacac atatattcgc aagatgtggc gtgttacggt
     7681 gaaaacctgg cctatttccc taaagggttt attgagaata tgtttttcgt gtcagccaat
     7741 ccctgggtga gtttcaccag ttttgattta aacgtggcca atatggacaa cttcttcgcc
     7801 cccgttttca ccatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg
     7861 attcaggttc atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct taatgaatta
     7921 caacagtact gcgatgagtg gcagggcggg gcgtaaacgc gtggcacata tacctgccgt
     7981 tcactattat ttagtgaaat gagatattat gatattttct gaattgtgat taaaaaggca
     8041 actttatgcc catgcaacag aaactataaa aaatacagag aatgaaaaga aacagataga
     8101 ttttttagtt ctttaggccc gtagtctgca aatcctttta tgattttcta tcaaacaaaa
     8161 gaggaaaata gaccagttgc aatccaaacg agagtctaat agaatgaggt cgaaaagtaa
     8221 atcgcgcggg tttgttactg ataaagcagg caagacctaa aatgtgtaaa gggcaaagtg
     8281 tatactttgg cgtcacccct tacatatttt aggtcttttt ttattgtgcg taactaactt
     8341 gccatcttca aacaggaggg ctggaagaag cagaccgcta acacagtaca taaaaaagga
     8401 gacatgaacg atgaacatca aaaagtttgc aaaacaagca acagtattaa cctttactac
     8461 cgcactgctg gcaggaggcg caactcaagc gtttgcgaaa gaaacgaacc aaaagccata
     8521 taaggaaaca tacggcattt cccatattac acgccatgat atgctgcaaa tccctgaaca
     8581 gcaaaaaaat gaaaaatatc aagttcctga attcgattcg tccacaatta aaaatatctc
     8641 ttctgcaaaa ggcctggacg tttgggacag ctggccatta caaaacgctg acggcactgt
     8701 cgcaaactat cacggctacc acatcgtctt tgcattagcc ggagatccta aaaatgcgga
     8761 tgacacatcg atttacatgt tctatcaaaa agtcggcgaa acttctattg acagctggaa
     8821 aaacgctggc cgcgtcttta aagacagcga caaattcgat gcaaatgatt ctatcctaaa
     8881 agaccaaaca caagaatggt caggttcagc cacatttaca tctgacggaa aaatccgttt
     8941 attctacact gatttctccg gtaaacatta cggcaaacaa acactgacaa ctgcacaagt
     9001 taacgtatca gcatcagaca gctctttgaa catcaacggt gtagaggatt ataaatcaat
     9061 ctttgacggt gacggaaaaa cgtatcaaaa tgtacagcag ttcatcgatg aaggcaacta
     9121 cagctcaggc gacaaccata cgctgagaga tcctcactac gtagaagata aaggccacaa
     9181 atacttagta tttgaagcaa acactggaac tgaagatggc taccaaggcg aagaatcttt
     9241 atttaacaaa gcatactatg gcaaaagcac atcattcttc cgtcaagaaa gtcaaaaact
     9301 tctgcaaagc gataaaaaac gcacggctga gttagcaaac ggcgctctcg gtatgattga
     9361 gctaaacgat gattacacac tgaaaaaagt gatgaaaccg ctgattgcat ctaacacagt
     9421 aacagatgaa attgaacgcg cgaacgtctt taaaatgaac ggcaaatggt acctgttcac
     9481 tgactcccgc ggatcaaaaa tgacgattga cggcattacg tctaacgata tttacatgct
     9541 tggttatgtt tctaattctt taactggccc atacaagccg ctgaacaaaa ctggccttgt
     9601 gttaaaaatg gatcttgatc ctaacgatgt aacctttact tactcacact tcgctgtacc
     9661 tcaagcgaaa ggaaacaatg tcgtgattac aagctatatg acaaacagag gattctacgc
     9721 agacaaacaa tcaacgtttg cgccaagctt cctgctgaac atcaaaggca agaaaacatc
     9781 tgttgtcaaa gacagcatcc ttgaacaagg acaattaaca gttaacaaat aaatgtcagg
     9841 ctcccttata cacagccagt ctgcacgtcg catagtgact ggatatgttg tgttttacag
     9901 tattatgtag tctgtttttt atgcaaaatc taatttaata tattgatatt tatatcattt
     9961 tacgtttctc gttcaacttt ttaatacaaa gttgcctccg gaacataatg gtgcatctag
    10021 atgcccattc ctgtagactt cttaattaag acgtcagaat tctcgaggcg gccgcatgtg
    10081 agtctcccta tagtgagtcg tattaatttc gcgggcgcgt cagcgggaga gccgttgtaa
    10141 ggcggca
//
