LOCUS       Exported                5813 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  FX sequencing vector for subcloning, kanamycin marker.
ACCESSION   .
VERSION     .
KEYWORDS    pINIT_kan
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5813)
  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 5813)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5813
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             complement(49..354)
                     /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(151..170)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(696..1355)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1270..1289
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1409..1439)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(1464..1588)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1607..1627)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(1631..1651)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      1781..1808
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(1880..2695)
                     /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     2603..2622
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2687..2706
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2817..3405
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3306..3325
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3559..3576
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3657..3676)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3733..4178
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             4179..5600
                     /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      5674..5760
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gctcttctag tcgactgcag actggctgtg tataagggag cctgacattt atattcccca
       61 gaacatcagg ttaatggcgt ttttgatgtc attttcgcgg tggctgagat cagccacttc
      121 ttccccgata acggagaccg gcacactggc catatcggtg gtcatcatgc gccagctttc
      181 atccccgata tgcaccaccg ggtaaagttc acgggagact ttatctgaca gcagacgtgc
      241 actggccagg gggatcacca tccgtcgccc gggcgtgtca ataatatcac tctgtacatc
      301 cacaaacaga cgataacggc tctctctttt ataggtgtaa accttaaact gcatttcacc
      361 agcccctgtt ctcgtcagca aaagagccgt tcatttcaat aaaccgggcg acctcagcca
      421 tcccttcctg attttccgct ttccagcgtt cggcacgcag acgacgggct tcattctgca
      481 tggttgtgct taccagaccg gagatattga catcatatat gccttgagca actgatagct
      541 gtcgctgtca actgtcactg taatacgctg cttcatagca tacctctttt tgacatactt
      601 cgggtataca tatcagtata tattcttata ccgcaaaaat cagcgcgcaa atacgcatac
      661 tgttatctgg cttttagtaa gccggatcca cgcgtttacg ccccgccctg ccactcatcg
      721 cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca aacggcatga
      781 tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccatg
      841 gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa actggtgaaa
      901 ctcacccagg gattggctga gacgaaaaac atattctcaa taaacccttt agggaaatag
      961 gccaggtttt caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa
     1021 tcgtcgtggt attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg
     1081 taacaagggt gaacactatc ccatatcacc agctcaccgt ctttcattgc catacggaat
     1141 tccggatgag cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc
     1201 ttatttttct ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag
     1261 gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata
     1321 tcaacggtgg tatatccagt gatttttttc tccattttag cttccttagc tcctgaaaat
     1381 ctcgacggat cctaactcaa aatccacaca ttatacgagc cggaagcata aagtgtaaag
     1441 cctggggtgc ctaatgcggc cgccatagtg actggatatg ttgtgtttta cagtattatg
     1501 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt
     1561 ctcgttcagc ttttttgtac aaacttgtga tatcgaattc gctagctacc tttctcttct
     1621 tttttggatc tacctttctc ttcttttttg gggatccact agttctgctc gcatgaagag
     1681 cggccaccga ggccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg
     1741 gcaggacgcc cgccataaac tgccaggcat caaattaagc agaaggccat cctgacggat
     1801 ggcctttttg cgtttctaca aactcttttg tttatttttc taaatacatt caaatatgta
     1861 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
     1921 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     1981 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     2041 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     2101 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     2161 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     2221 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     2281 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     2341 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     2401 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     2461 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     2521 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     2581 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     2641 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     2701 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     2761 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     2821 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     2881 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     2941 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     3001 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     3061 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     3121 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     3181 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     3241 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     3301 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     3361 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     3421 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     3481 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     3541 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cgcaagagcg cctgatgcgg
     3601 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca
     3661 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     3721 gcccagccgg cccacatata cctgccgttc actattattt agtgaaatga gatattatga
     3781 tattttctga attgtgatta aaaaggcaac tttatgccca tgcaacagaa actataaaaa
     3841 atacagagaa tgaaaagaaa cagatagatt ttttagttct ttaggcccgt agtctgcaaa
     3901 tccttttatg attttctatc aaacaaaaga ggaaaataga ccagttgcaa tccaaacgag
     3961 agtctaatag aatgaggtcg aaaagtaaat cgcgcgggtt tgttactgat aaagcaggca
     4021 agacctaaaa tgtgtaaagg gcaaagtgta tactttggcg tcacccctta catattttag
     4081 gtcttttttt attgtgcgta actaacttgc catcttcaaa caggagggct ggaagaagca
     4141 gaccgctaac acagtacata aaaaaggaga catgaacgat gaacatcaaa aagtttgcaa
     4201 aacaagcaac agtattaacc tttactaccg cactgctggc aggaggcgca actcaagcgt
     4261 ttgcgaaaga aacgaaccaa aagccatata aggaaacata cggcatttcc catattacac
     4321 gccatgatat gctgcaaatc cctgaacagc aaaaaaatga aaaatataaa gttcctgagt
     4381 tcgattcgtc cacaattaaa aatatctctt ctgcaaaagg cctggacgtt tgggacagct
     4441 ggccattaca aaacactgac ggcactgtcg caaactatca cggctaccac atcgtctttg
     4501 cattagccgg agatcctaaa aatgcggatg acacatcgat ttacatgttc tatcaaaaag
     4561 tcggcgaaac ttctattgac agctggaaaa acgctggccg cgtctttaaa gacagcgaca
     4621 aattcgatgc aaatgattct atcctaaaag accaaacaca agaatggtca ggttcagcca
     4681 catttacatc tgacggaaaa atccgtttat tctacactga tttctccggt aaacattacg
     4741 gcaaacaaac actgacaact gcacaagtta acgtatcagc atcagacagc tctttgaaca
     4801 tcaacggtgt agaggattat aaatcaatct ttgacggtga cggaaaaacg tatcaaaatg
     4861 tacagcagtt catcgatgaa ggcaactaca gctcaggcga caaccatacg ctgagagatc
     4921 ctcactacgt agaagataaa ggccacaaat acttagtatt tgaagcaaac actggaactg
     4981 aagatggcta ccaaggcgaa gaatctttat ttaacaaagc atactatggc aaaagcacat
     5041 cattcttccg tcaagaaagt caaaaacttc tgcaaagcga taaaaaacgc acggctgagt
     5101 tagcaaacgg cgctctcggt atgattgagc taaacgatga ttacacactg aaaaaagtga
     5161 tgaaaccgct gattgcatct aacacagtaa cagatgaaat tgaacgcgcg aacgtcttta
     5221 aaatgaacgg caaatggtac ctgttcactg actcccgcgg atcaaaaatg acgattgacg
     5281 gcattacgtc taacgatatt tacatgcttg gttatgtttc taattcttta actggcccat
     5341 acaagccgct gaacaaaact ggccttgtgt taaaaatgga tcttgatcct aacgatgtaa
     5401 cctttactta ctcacacttc gctgtacctc aagcgaaagg aaacaatgtc gtgattacaa
     5461 gctatatgac aaacagagga ttctacgcag acaaacaatc aacgtttgcg cctagcttcc
     5521 tgctgaacat caaaggcaag aaaacatctg ttgtcaaaga cagcatcctt gaacaaggac
     5581 aattaacagt taacaaataa aaacgcaaaa gaaaatgccg atatcctatt ggcattgcgg
     5641 ccgcggtctc ggcggtgggt cgtctagagg catcaaataa aacgaaaggc tcagtcgaaa
     5701 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
     5761 ccgccgccct agacctagcg gccggccgca agcgctgcac ggcctggttg gcc
//
