LOCUS       pGG-F-T7-pT7-RBS-A-sacB-linker-G-G        4807 bp    DNA     circular     21-APR-2023
DEFINITION  .
ACCESSION   p00626
KEYWORDS    .
SOURCE      
  ORGANISM  .
FEATURES             Location/Qualifiers
     GG              1..11
                     /modified_by="wacae"
                     /note="Derived using Geneious Prime 2022.0.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="F"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:8c-dgycryy"">F</a>"
                     /label="F"
     misc_feature    12..140
                     /Original_Bases
                     /label
                     /note="Geneious type: Editing History Insertion"
     terminator      12..140
                     /created_by="mamee"
                     /label="T7 Transcription Terminator"
     promoter        141..158
                     /created_by="mamee"
                     /modified_by="mamee"
                     /label="T7 Promoter"
     misc_feature    144..214
                     /Original_Bases="GCTAGGTAGTAACTAGT"
                     /label="GCTAGGTAGTAACTAGT"
                     /note="Geneious type: Editing History Replacement"
     RBS             206..212
                     /created_by="mamee"
     GG              215..218
                     /created_by="mamee"
                     /modified_by="mamee"
                     /label="A"
     promoter        230..675
                     /gene="sacR"
                     /note="sacB promoter and control region"
                     /label="sacB promoter"
     primer_bind     550..569
                     /%GC="55.0"
                     /Hairpin_Tm="37.8"
                     /Self_Dimer_Tm="None"
                     /Tm="60.3"
                     /annotation_group="6171301720505005faaec39-ee7b-4a93-b4db-
                     347313541848"
                     /created_by="primer3"
                     /Sequence="CTTTGGCGTCACCCCTTACA"
                     /Mismatches=0
                     /#_Off-target_Sites=0
                     /Off-target_Template="pGG-F-T7-pT7-RBS-A-sacB-linker-G-G"
                     /label="MAMEE244"
     CDS             676..2097
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /note="negative selection marker"
                     /Original_Translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQK
                     PYKETYGISHITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNA
                     DGTVANYHGYHIVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDA
                     NDSILKDQTQEWSGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNIN
                     GVEDYKSIFDGDGKTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGT
                     EDGYQGEESLFNKAYYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLK
                     KVMKPLIASNTVTDEIERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNS
                     LTGPYKPLNKTGLVLKMDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQS
                     TFAPSFLLNIKGKKTSVVKDSILEQGQLTVNK"
                     /label="SacB"
     primer_bind     1356..1376
                     /%GC="42.9"
                     /Hairpin_Tm="34.4"
                     /Self_Dimer_Tm="24.0"
                     /Tm="57.1"
                     /annotation_group="617130261740200a9d8c2da-a922-409a-85da-
                     c68d99780a82"
                     /created_by="primer3"
                     /Sequence="TGTACAGCAGTTCATCGATGA"
                     /Mismatches=0
                     /#_Off-target_Sites=0
                     /Off-target_Template="pGG-F-T7-pT7-RBS-A-sacB-linker-G-G"
                     /label="MAMEE245"
     GG              2109..2112
                     /created_by="mamee"
                     /label="G"
     GG              complement(2133..2143)
                     /created_by="thjac"
                     /note="Derived using Geneious Prime 2022.0.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="G"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:84-dgycryy"">G</a>"
                     /label="G"
     primer          complement(2163..2183)
                     /note="Derived using Geneious Prime 2022.0.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="pGG-B-HA-TurboID-linker-C - GG_SEQ_rev"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0;
                     GG_SEQ_rev (pGG-B-HA-TurboID-linker-C - GG_SEQ_rev)"
                     /label="GG_SEQ_rev"
     primer_bind     complement(2288..2307)
                     /%GC="55.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Tm="60.3"
                     /annotation_group="617130083026600040f805d-174c-4433-825a-
                     463b500d5fce"
                     /created_by="primer3"
                     /Sequence="AACTGTTGGGAAGGGCGATC"
                     /Mismatches=0
                     /#_Off-target_Sites=0
                     /Off-target_Template="pGG-F-T7-pT7-RBS-A-sacB-linker-G-G"
                     /label="MAMEE242"
     promoter        2663..2767
                     /created_by="mamee"
                     /modified_by="mamee"
                     /note="Derived using Geneious Prime 2023.0.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="AmpR"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; amp (<a
                     href=""urn:local:.:8i-dgycryy"">AmpR</a>)"
                     /label="amp"
     misc_feature    2768..3628
                     /X-Transferred_From="AmpR"
                     /X-Transferred_Similarity="100.00%"
                     /modified_by="mamee"
                     /note="Derived using Geneious Prime 2023.0.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="AmpR"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:8i-dgycryy"">AmpR</a>"
                     /label="AmpR"
                     /note="Geneious type: Bacterial Selection"
     rep_origin      3799..4387
                     /created_by="thjac"
                     /modified_by="mamee"
                     /note="Derived using Geneious Prime 2022.0.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="high-copy-number ColE1/pMB1/pBR322/pUC
                     origin of  replication"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0;
                     ColE1/pMB1/pBR322/pUC Ori (<a
                     href=""urn:local:.:8d-dk4mzop"">high-copy-number
                     ColE1/pMB1/pBR322/pUC origin of  replication</a>)"
                     /label="ColE1/pMB1/pBR322/pUC Ori"
     primer_bind     4599..4618
                     /%GC="50.0"
                     /Hairpin_Tm="39.6"
                     /Self_Dimer_Tm="None"
                     /Tm="59.6"
                     /annotation_group="617129578217900b8b36bfe-70d3-41db-b28a-
                     9f804061c2d1"
                     /created_by="primer3"
                     /Sequence="CGCGTTGGCCGATTCATTAA"
                     /Mismatches=0
                     /#_Off-target_Sites=0
                     /Off-target_Template="pGG-F-T7-pT7-RBS-A-sacB-linker-G-G"
                     /label="MAMEE156"
     primer          4751..4772
                     /note="Derived using Geneious Prime 2022.0.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="pGG-B-HA-TurboID-linker-C - GG_SEQ_fw"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0;
                     GG_SEQ_fw (pGG-B-HA-TurboID-linker-C - GG_SEQ_fw)"
                     /label="GG_SEQ_fw"
ORIGIN      
        1 ggtctcaact actgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
       61 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
      121 aggaggaact atatccggat taatacgact cactataggg agtccacaac ggtttccctc
      181 tagaaataat tttgtttaac tttaagaagg agtcacctgg atgcaggtgc acatatacct
      241 gccgttcact attatttagt gaaatgagat attatgatat tttctgaatt gtgattaaaa
      301 aggcaacttt atgcccatgc aacagaaact ataaaaaata cagagaatga aaagaaacag
      361 atagattttt tagttcttta ggcccgtagt ctgcaaatcc ttttatgatt ttctatcaaa
      421 caaaagagga aaatagacca gttgcaatcc aaacgagagt ctaatagaat gaggtcgaaa
      481 agtaaatcgc gcgggtttgt tactgataaa gcaggcaaga cctaaaatgt gtaaagggca
      541 aagtgtatac tttggcgtca ccccttacat attttaggtc tttttttatt gtgcgtaact
      601 aacttgccat cttcaaacag gagggctgga agaagcagac cgctaacaca gtacataaaa
      661 aaggagacat gaacgatgaa catcaaaaag tttgcaaaac aagcaacagt attaaccttt
      721 actaccgcac tgctggcagg aggcgcaact caagcgtttg cgaaagaaac gaaccaaaag
      781 ccatataagg aaacatacgg catttcccat attacacgcc atgatatgct gcaaatccct
      841 gaacagcaaa aaaatgaaaa atatcaagtt cctgagttcg attcgtccac aattaaaaat
      901 atctcttctg caaaaggcct ggacgtttgg gacagctggc cattacaaaa cgctgacggc
      961 actgtcgcaa actatcacgg ctaccacatc gtctttgcat tagccggaga tcctaaaaat
     1021 gcggatgaca catcgattta catgttctat caaaaagtcg gcgaaacttc tattgacagc
     1081 tggaaaaacg ctggccgcgt ctttaaagac agcgacaaat tcgatgcaaa tgattctatc
     1141 ctaaaagacc aaacacaaga atggtcaggt tcagccacat ttacatctga cggaaaaatc
     1201 cgtttattct acactgattt ctccggtaaa cattacggca aacaaacact gacaactgca
     1261 caagttaacg tatcagcatc agacagctct ttgaacatca acggtgtaga ggattataaa
     1321 tcaatctttg acggtgacgg aaaaacgtat caaaatgtac agcagttcat cgatgaaggc
     1381 aactacagct caggcgacaa ccatacgctg agagatcctc actacgtaga agataaaggc
     1441 cacaaatact tagtatttga agcaaacact ggaactgaag atggctacca aggcgaagaa
     1501 tctttattta acaaagcata ctatggcaaa agcacatcat tcttccgtca agaaagtcaa
     1561 aaacttctgc aaagcgataa aaaacgcacg gctgagttag caaacggcgc tctcggtatg
     1621 attgagctaa acgatgatta cacactgaaa aaagtgatga aaccgctgat tgcatctaac
     1681 acagtaacag atgaaattga acgcgcgaac gtctttaaaa tgaacggcaa atggtacctg
     1741 ttcactgact cccgcggatc aaaaatgacg attgacggca ttacgtctaa cgatatttac
     1801 atgcttggtt atgtttctaa ttctttaact ggcccataca agccgctgaa caaaactggc
     1861 cttgtgttaa aaatggatct tgatcctaac gatgtaacct ttacttactc acacttcgct
     1921 gtacctcaag cgaaaggaaa caatgtcgtg attacaagct atatgacaaa cagaggattc
     1981 tacgcagaca aacaatcaac gtttgcgccg agcttcctgc tgaacatcaa aggcaagaaa
     2041 acatctgttg tcaaagacag catccttgaa caaggacaat taacagttaa caaataacac
     2101 ctgcagctgt atctagaatc gcgtatgcga gtgtattgag accgaattct cgccctatag
     2161 tgagtcgtat tacctggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     2221 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     2281 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg
     2341 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac
     2401 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     2461 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     2521 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct
     2581 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     2641 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     2701 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     2761 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     2821 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     2881 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     2941 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     3001 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     3061 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     3121 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     3181 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     3241 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     3301 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     3361 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     3421 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     3481 tggctctcga ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     3541 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     3601 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     3661 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     3721 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     3781 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     3841 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     3901 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     3961 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     4021 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     4081 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     4141 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     4201 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     4261 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     4321 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     4381 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     4441 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     4501 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     4561 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     4621 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     4681 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
     4741 gtgtggaatt gtgagcggat aacaatttca cacgagctat ttaggtgaca ctatagaagt
     4801 gaagctt
//
