LOCUS       sequence-251594-        6032 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Destination vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6032)
  AUTHORS   Diebold-Durand ML, Burmann F, Gruber S
  TITLE     High-Throughput Allelic Replacement Screening in Bacillus subtilis.
  JOURNAL   Methods Mol Biol. 2019;2004:49-61. doi: 10.1007/978-1-4939-9520-2_5.
  PUBMED    31147909
REFERENCE   2  (bases 1 to 6032)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Methods Mol
            Biol."; date: "2019"; volume: "2004:49-61. doi"; pages: " 
            10.1007/978-1-4939-9520-2_5"
COMMENT     Sequence Label: pET-Gate2 ccdB
FEATURES             Location/Qualifiers
     source          1..6032
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     protein_bind    90..106
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     95..117
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     114..130
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     114..130
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     RBS             215..223
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             225..533
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MIQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSD
                     KVSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     412..431
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     misc_feature    532..588
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(589..606)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(589..605)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(598..620)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(967..985)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      981..1028
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1065..1520
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1152..1171)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1362..1383
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1612..2427)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1612..2427)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2335..2354
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2419..2438
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2549..3137
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3038..3057
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3291..3308
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3323..3462
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3548..3570
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3564..3755)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4564..5646)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5608..5627
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5647..5724)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5930..5949
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gtctcgagtg
       61 agaccttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
      121 cagctatgac catgattacg ccaagcttga tatcgaattc ctgcagccca aactgcagtt
      181 gacaacataa aaactttgtg ttatacttgt aacgtaagga ggtaatgatt cagtttaagg
      241 tttacaccta taaaagagag agccgttatc gtctgtttgt ggatgtacag agtgatatta
      301 ttgacacgcc cgggcgacgg atggtgatcc ccctggccag tgcacgtctg ctgtcagata
      361 aagtctcccg tgaactttac ccggtggtgc atatcgggga tgaaagctgg cgcatgatga
      421 ccaccgatat ggccagtgtg ccggtctccg ttatcgggga agaagtggct gatctcagcc
      481 accgcgaaaa tgacatcaaa aacgccatta acctgatgtt ctggggaata taagcttgca
      541 tgcctgcagg tcgactctag aggatccccg ggtaccgagc tcgaattcac tggccgtcgt
      601 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
      661 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
      721 gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg
      781 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
      841 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
      901 gggtctctcc atggtgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
      961 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     1021 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     1081 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     1141 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     1201 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     1261 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     1321 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     1381 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     1441 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     1501 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     1561 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa
     1621 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     1681 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     1741 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
     1801 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     1861 tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     1921 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     1981 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     2041 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     2101 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     2161 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     2221 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     2281 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     2341 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca
     2401 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga
     2461 cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     2521 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     2581 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     2641 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     2701 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     2761 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     2821 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     2881 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     2941 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3001 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     3061 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     3121 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3181 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     3241 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     3301 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     3361 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3421 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     3481 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     3541 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     3601 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     3661 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     3721 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     3781 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     3841 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     3901 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     3961 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     4021 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     4081 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     4141 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     4201 agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg
     4261 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     4321 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     4381 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     4441 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     4501 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     4561 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4621 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     4681 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     4741 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     4801 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     4861 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     4921 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     4981 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     5041 atgccagcca gccagacgca gacgcgccga gacagaactt aatggtcccg ctaacagcgc
     5101 gatttgctga tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     5161 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     5221 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     5281 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     5341 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     5401 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     5461 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     5521 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     5581 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     5641 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     5701 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     5761 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
     5821 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
     5881 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     5941 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     6001 cgtagaggat cgagatctcg atcccgcgaa at
//
