LOCUS       sequence-66904-e        5599 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FX sequencing vector for subcloning, tetracycline marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5599)
  AUTHORS   Geertsma ER, Dutzler R
  TITLE     A versatile and efficient high-throughput cloning tool for 
            structural biology.
  JOURNAL   Biochemistry. 2011 Apr 19;50(15):3272-8. Epub 2011 Mar 25.
  PUBMED    21410291
REFERENCE   2  (bases 1 to 5599)
  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: 
            "Biochemistry."; date: "2011-04-19"; volume: "50(15)"; pages: 
            "3272-8. Epub 2011 Mar 25"
COMMENT     Sequence Label: pINIT_tet
FEATURES             Location/Qualifiers
     source          1..5599
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(55..360)
                     /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="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(157..176)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     terminator      785..812
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(1383..2573)
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSITGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     2202..2221
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        complement(join(2621..2626,2627..2643,2644..2649))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     rep_origin      2737..3325
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3226..3245
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3479..3496
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3524..3969
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3970..4056,4057..5391)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYKVPEFDSSTIKNISSAKGLDVWDSWPLQNTDGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     terminator      5465..5551
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 tggccgctct tctagtcgac ctgcagactg gctgtgtata agggagcctg acatttatat
       61 tccccagaac atcaggttaa tggcgttttt gatgtcattt tcgcggtggc tgagatcagc
      121 cacttcttcc ccgataacgg agaccggcac actggccata tcggtggtca tcatgcgcca
      181 gctttcatcc ccgatatgca ccaccgggta aagttcacgg gagactttat ctgacagcag
      241 acgtgcactg gccaggggga tcaccatccg tcgcccgggc gtgtcaataa tatcactctg
      301 tacatccaca aacagacgat aacggctctc tcttttatag gtgtaaacct taaactgcat
      361 ttcaccagcc cctgttctcg tcagcaaaag agccgttcat ttcaataaac cgggcgacct
      421 cagccatccc ttcctgattt tccgctttcc agcgttcggc acgcagacga cgggcttcat
      481 tctgcatggt tgtgcttacc agaccggaga tattgacatc atatatgcct tgagcaactg
      541 atagctgtcg ctgtcaactg tcactgtaat acgctgcttc atagcatacc tctttttgac
      601 atacttcggg tatacatatc agtatatatt cttataccgc aaaaatcagc gcgcaaatac
      661 gcatactgtt atctgcatga agagcggcca ccgaggccgg gagcggattt gaacgttgcg
      721 aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt
      781 aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt
      841 tttctaaata cattcaaata tgtatccgct catggagcta ccggacagcg gtgcggactg
      901 ttgtaactca gaataagaaa tgaggccgct catggcgttg actctcagtc atagtatcgt
      961 ggtatcaccg gttggttcca ctctctgttg cgggcaactt cagcagcacg taggggactt
     1021 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     1081 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     1141 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     1201 gcgcacccgt ggccaggacc caacgctgcc cgagatgcgc cgcgtgcggc tgctggagat
     1261 ggcggacgcg atggatatgt tctgccaagg gttggtttgc gcattcacag ttctccgcaa
     1321 gaattgattg gctccaattc ttggagtggt gaatccgtta gcgaggtgcc gccggcttcc
     1381 attcaggtcg aggtggcccg gctccatgca ccgcgacgca acgcggggag gcagacaagg
     1441 tatagggcgg cgcctacaat ccatgccaac ccgttccatg tgctcgccga ggcggcataa
     1501 atcgccgtga cgatcagcgg tccagtgatc gaagttaggc tggtaagagc cgcgagcgat
     1561 ccttgaagct gtccctgatg gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg
     1621 ggcatcccga tgccgccgga agcgagaaga atcataatgg ggaaggccat ccagcctcgc
     1681 gtcgcgaacg ccagcaagac gtagcccagc gcgtcggccg ccatgccggc gataatggcc
     1741 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     1801 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     1861 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     1921 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     1981 ttgaaggctc tcaagggcat cggtcgacgc tctcccttat gcgactcctg cattaggaag
     2041 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     2101 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     2161 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     2221 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     2281 atccacagga cgggtgtggt cgccatgatc gcgtagtcga tagtggctcc aagtagcgaa
     2341 gcgagcagga ctgggcggcg gccaaagcgg tcggacagtg ctccgagaac gggtgcgcat
     2401 agaaattgca tcaacgcata tagcgctagc agcacgccat agtgactggc gatgctgtcg
     2461 gaatggacga tatcccgcaa gaggcccggc agtaccggca taaccaagcc tatgcctaca
     2521 gcatccaggg tgacggtgcc gaggatgacg atgagcgcat tgttagattt catacacggt
     2581 gcctgactgc gttagcaatt taactgtgat aaactaccgc attaaagctt atcgatgata
     2641 agctgtcaaa catgagaatt ccatgaccaa aatcccttaa cgtgagtttt cgttccactg
     2701 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     2761 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     2821 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     2881 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     2941 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3001 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3061 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3121 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3181 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3241 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3301 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3361 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3421 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3481 cgagtcagtg agcgaggaag cggaagatcg ggcccagccg gcccacatat acctgccgtt
     3541 cactattatt tagtgaaatg agatattatg atattttctg aattgtgatt aaaaaggcaa
     3601 ctttatgccc atgcaacaga aactataaaa aatacagaga atgaaaagaa acagatagat
     3661 tttttagttc tttaggcccg tagtctgcaa atccttttat gattttctat caaacaaaag
     3721 aggaaaatag accagttgca atccaaacga gagtctaata gaatgaggtc gaaaagtaaa
     3781 tcgcgcgggt ttgttactga taaagcaggc aagacctaaa atgtgtaaag ggcaaagtgt
     3841 atactttggc gtcacccctt acatatttta ggtctttttt tattgtgcgt aactaacttg
     3901 ccatcttcaa acaggagggc tggaagaagc agaccgctaa cacagtacat aaaaaaggag
     3961 acatgaacga tgaacatcaa aaagtttgca aaacaagcaa cagtattaac ctttactacc
     4021 gcactgctgg caggaggcgc aactcaagcg tttgcgaaag aaacgaacca aaagccatat
     4081 aaggaaacat acggcatttc ccatattaca cgccatgata tgctgcaaat ccctgaacag
     4141 caaaaaaatg aaaaatataa agttcctgag ttcgattcgt ccacaattaa aaatatctct
     4201 tctgcaaaag gcctggacgt ttgggacagc tggccattac aaaacactga cggcactgtc
     4261 gcaaactatc acggctacca catcgtcttt gcattagccg gagatcctaa aaatgcggat
     4321 gacacatcga tttacatgtt ctatcaaaaa gtcggcgaaa cttctattga cagctggaaa
     4381 aacgctggcc gcgtctttaa agacagcgac aaattcgatg caaatgattc tatcctaaaa
     4441 gaccaaacac aagaatggtc aggttcagcc acatttacat ctgacggaaa aatccgttta
     4501 ttctacactg atttctccgg taaacattac ggcaaacaaa cactgacaac tgcacaagtt
     4561 aacgtatcag catcagacag ctctttgaac atcaacggtg tagaggatta taaatcaatc
     4621 tttgacggtg acggaaaaac gtatcaaaat gtacagcagt tcatcgatga aggcaactac
     4681 agctcaggcg acaaccatac gctgagagat cctcactacg tagaagataa aggccacaaa
     4741 tacttagtat ttgaagcaaa cactggaact gaagatggct accaaggcga agaatcttta
     4801 tttaacaaag catactatgg caaaagcaca tcattcttcc gtcaagaaag tcaaaaactt
     4861 ctgcaaagcg ataaaaaacg cacggctgag ttagcaaacg gcgctctcgg tatgattgag
     4921 ctaaacgatg attacacact gaaaaaagtg atgaaaccgc tgattgcatc taacacagta
     4981 acagatgaaa ttgaacgcgc gaacgtcttt aaaatgaacg gcaaatggta cctgttcact
     5041 gactcccgcg gatcaaaaat gacgattgac ggcattacgt ctaacgatat ttacatgctt
     5101 ggttatgttt ctaattcttt aactggccca tacaagccgc tgaacaaaac tggccttgtg
     5161 ttaaaaatgg atcttgatcc taacgatgta acctttactt actcacactt cgctgtacct
     5221 caagcgaaag gaaacaatgt cgtgattaca agctatatga caaacagagg attctacgca
     5281 gacaaacaat caacgtttgc gcctagcttc ctgctgaaca tcaaaggcaa gaaaacatct
     5341 gttgtcaaag acagcatcct tgaacaagga caattaacag ttaacaaata aaaacgcaaa
     5401 agaaaatgcc gatatcctat tggcattgcg gccgcggtct cggcggtggg tcgtctagag
     5461 gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt
     5521 gtcggtgaac gctctcctga gtaggacaaa tccgccgccc tagacctagc ggccggccgc
     5581 aagcgctgca cggcctggt
//
