LOCUS       sequence-66358-e        4980 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FX sequencing vector for subcloning, chloramphenicol marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4980)
  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 4980)
  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_cat
FEATURES             Location/Qualifiers
     source          1..4980
                     /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"
     promoter        1010..1112
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1113..1772
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1179..1198)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      2118..2706
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2607..2626
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2860..2877
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        2905..3350
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3351..3437,3438..4772)
                     /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      4846..4932
                     /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 catgaattcc ccgcgcgcga tgccctttcg
      901 tcttcgaata aatacctgtg acggaagatc acttcgcaga ataaataaat cctggtgtcc
      961 ctgttgatac cgggaagccc tgggccaact tttggcgaaa atgagacgtt gatcggcacg
     1021 taagaggttc caactttcac cataatgaaa taagatcact accgggcgta ttttttgagt
     1081 tatcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact ggatatacca
     1141 ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag tcagttgctc
     1201 aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag accgtaaaga
     1261 aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg atgaatgctc
     1321 atccggaatt tcgtatggca atgaaagacg gtgagctggt gatatgggat agtgttcacc
     1381 cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg agtgaatacc
     1441 acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa
     1501 acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca gccaatccct
     1561 gggtgagttt caccagtttt gatttaaacg tggcgaatat ggacaacttc ttcgcccccg
     1621 ttttcactat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc
     1681 aggttcatca tgccgtctgt gatggcttcc atgtcggcag aatgcttaat gaattacaac
     1741 agtactgcga tgagtggcag ggcggggcgt aattttttta aggcagttat tggtgccctt
     1801 aaacgcctgg ttgctacgcc tgaataagtg ataataagcg gatgaatggc agaaattcga
     1861 gcccgcctaa tgagcgggct tttttttagc ccgcctaatg agcgggcttt tttttcgaaa
     1921 gcaaattcga cccatcgcgc gcggggagtc aactcagcaa aagttcgatt tattcaacaa
     1981 agccacgttg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca
     2041 tgcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     2101 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     2161 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     2221 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     2281 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     2341 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     2401 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     2461 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     2521 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     2581 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     2641 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     2701 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     2761 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     2821 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     2881 gcggaagatc gggcccagcc ggcccacata tacctgccgt tcactattat ttagtgaaat
     2941 gagatattat gatattttct gaattgtgat taaaaaggca actttatgcc catgcaacag
     3001 aaactataaa aaatacagag aatgaaaaga aacagataga ttttttagtt ctttaggccc
     3061 gtagtctgca aatcctttta tgattttcta tcaaacaaaa gaggaaaata gaccagttgc
     3121 aatccaaacg agagtctaat agaatgaggt cgaaaagtaa atcgcgcggg tttgttactg
     3181 ataaagcagg caagacctaa aatgtgtaaa gggcaaagtg tatactttgg cgtcacccct
     3241 tacatatttt aggtcttttt ttattgtgcg taactaactt gccatcttca aacaggaggg
     3301 ctggaagaag cagaccgcta acacagtaca taaaaaagga gacatgaacg atgaacatca
     3361 aaaagtttgc aaaacaagca acagtattaa cctttactac cgcactgctg gcaggaggcg
     3421 caactcaagc gtttgcgaaa gaaacgaacc aaaagccata taaggaaaca tacggcattt
     3481 cccatattac acgccatgat atgctgcaaa tccctgaaca gcaaaaaaat gaaaaatata
     3541 aagttcctga gttcgattcg tccacaatta aaaatatctc ttctgcaaaa ggcctggacg
     3601 tttgggacag ctggccatta caaaacactg acggcactgt cgcaaactat cacggctacc
     3661 acatcgtctt tgcattagcc ggagatccta aaaatgcgga tgacacatcg atttacatgt
     3721 tctatcaaaa agtcggcgaa acttctattg acagctggaa aaacgctggc cgcgtcttta
     3781 aagacagcga caaattcgat gcaaatgatt ctatcctaaa agaccaaaca caagaatggt
     3841 caggttcagc cacatttaca tctgacggaa aaatccgttt attctacact gatttctccg
     3901 gtaaacatta cggcaaacaa acactgacaa ctgcacaagt taacgtatca gcatcagaca
     3961 gctctttgaa catcaacggt gtagaggatt ataaatcaat ctttgacggt gacggaaaaa
     4021 cgtatcaaaa tgtacagcag ttcatcgatg aaggcaacta cagctcaggc gacaaccata
     4081 cgctgagaga tcctcactac gtagaagata aaggccacaa atacttagta tttgaagcaa
     4141 acactggaac tgaagatggc taccaaggcg aagaatcttt atttaacaaa gcatactatg
     4201 gcaaaagcac atcattcttc cgtcaagaaa gtcaaaaact tctgcaaagc gataaaaaac
     4261 gcacggctga gttagcaaac ggcgctctcg gtatgattga gctaaacgat gattacacac
     4321 tgaaaaaagt gatgaaaccg ctgattgcat ctaacacagt aacagatgaa attgaacgcg
     4381 cgaacgtctt taaaatgaac ggcaaatggt acctgttcac tgactcccgc ggatcaaaaa
     4441 tgacgattga cggcattacg tctaacgata tttacatgct tggttatgtt tctaattctt
     4501 taactggccc atacaagccg ctgaacaaaa ctggccttgt gttaaaaatg gatcttgatc
     4561 ctaacgatgt aacctttact tactcacact tcgctgtacc tcaagcgaaa ggaaacaatg
     4621 tcgtgattac aagctatatg acaaacagag gattctacgc agacaaacaa tcaacgtttg
     4681 cgcctagctt cctgctgaac atcaaaggca agaaaacatc tgttgtcaaa gacagcatcc
     4741 ttgaacaagg acaattaaca gttaacaaat aaaaacgcaa aagaaaatgc cgatatccta
     4801 ttggcattgc ggccgcggtc tcggcggtgg gtcgtctaga ggcatcaaat aaaacgaaag
     4861 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     4921 agtaggacaa atccgccgcc ctagacctag cggccggccg caagcgctgc acggcctggt
//
