LOCUS       Exported                5841 bp ds-DNA     circular SYN 10-SEP-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5841)
  AUTHORS   Marek Basler
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Jan 28, 2021 from SnapGene Viewer 5.2.4
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5841
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     1..32
                     /label=Fragment 1.FOR
     primer_bind     complement(9..40)
                     /label=Fragment 2.REV
     primer_bind     53..73
                     /label=oOPC-043
     primer_bind     120..139
                     /label=pUCM13-52
                     /note="Microsynth"
     primer_bind     152..168
                     /label=M13-40
                     /note="Microsynth"
     primer_bind     161..179
                     /label=BAC2
                     /note="Microsynth"
     primer_bind     171..187
                     /label=M13
                     /note="MicrosynthMicrosynth"
     primer_bind     198..216
                     /label=T7
                     /note="Microsynth"
     misc_feature    231..260
                     /label=I-SceI
     primer_bind     complement(433..450)
                     /label=oOPC-255
     CDS             complement(451..1245)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     781..801
                     /label=oOPC-044
     misc_feature    complement(1334..1383)
                     /label=Kan promoter
     primer_bind     1384..1406
                     /label=oOPC-254
     primer_bind     1448..1467
                     /label=oOPC-041
     primer_bind     1448..1467
                     /label=oOPC-041
     misc_feature    1471..1609
                     /label=Cloning cassette
     primer_bind     complement(1507..1531)
                     /label=oFRC332
     primer_bind     complement(1545..1561)
                     /label=SK
                     /note="Microsynth"
     primer_bind     1546..1567
                     /label=oFRC333
     primer_bind     complement(1599..1615)
                     /label=T3
                     /note="Microsynth"
     primer_bind     complement(1622..1641)
                     /label=oOPC-042
     primer_bind     complement(1622..1641)
                     /label=oOPC-042
     primer_bind     complement(1637..1654)
                     /label=M13r
                     /note="Microsynth"
     primer_bind     complement(1638..1654)
                     /label=M13r-short
                     /note="Microsynth"
     primer_bind     complement(1652..1671)
                     /label=LacOp-for
                     /note="Microsynth"
     primer_bind     complement(1664..1682)
                     /label=Pet-T7side
                     /note="Microsynth"
     primer_bind     complement(1686..1705)
                     /label=pUCM13-rev-57
                     /note="Microsynth"
     primer_bind     complement(1763..1780)
                     /label=oOPC-381
     primer_bind     1881..1896
                     /label=oOPC-380
     rep_origin      1954..2342
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     primer_bind     2186..2210
                     /label=oOPC-045
     primer_bind     complement(2345..2359)
                     /label=pET-up
                     /note="Microsynth"
     primer_bind     complement(2402..2426)
                     /label=oOPC-065
     CDS             complement(2427..3050)
                     /codon_start=1
                     /label=tetR
                     /note="tetracycline repressor derived from transposon Tn10"
                     /translation="MSRLDKSKVISSALELLNEVGIEGLTTRKLAQRLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     2737..2746
                     /label=pET-down
                     /note="Microsynth"
     promoter        3070..3123
                     /label=PtetA
                     /note="tetA promoter"
     -35_signal      3070..3075
                     /label=-35
                     /note="-35 tetA promoter from transposon Tn10"
     CDS             3085..3093
                     /label=TetR-bind
                     /note="site A TetR moiety"
     -10_signal      3094..3100
                     /label=-10
                     /note="-10 tetA promoter from transposon Tn10"
     CDS             3115..3123
                     /label=TetR-bind
                     /note="site B TetR moiety"
     CDS             3132..3833
                     /codon_start=1
                     /label=I-SceI
                     /note="I-SceI meganuclease"
                     /translation="MHQKNQVMNLGPNSKLLKEYKSQLIELNIEQFEAGIGLILGDAYI
                     RSRDEGKTYCMQFEWKNKAYMDHVCLLYDQWVLSPPHKKERVNHLGNLVITWGAQTFKH
                     QAFNKLANLFIVNNKKTIPNNLVENYLTPMSLAYWFMDDGGKWDYNKNSTNKSIVLNTQ
                     SFTFEEVEYLVKGLRNKFQLNCYVKINKNKPIIYIDSMSYLIFYNLIKPYLIPQMMYKL
                     PNTISSETFLK"
     primer_bind     3766..3785
                     /label=oOPC-047
     primer_bind     complement(3805..3839)
                     /label=oFRC27
     primer_bind     complement(3808..3859)
                     /label=oFRC57
     primer_bind     3820..3864
                     /label=oFRC58
     misc_feature    3840..5278
                     /label=Sac
     promoter        3840..3854
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             3855..5276
                     /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
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKPLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     primer_bind     complement(5242..5296)
                     /label=oFRC28
     primer_bind     5257..5300
                     /label=oFRC26
     primer_bind     complement(5286..5335)
                     /label=oOPC-016
     CDS             complement(5331..5702)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     primer_bind     5567..5576
                     /label=M13
                     /note="MicrosynthMicrosynth"
     oriT            join(5735..5841,1..3)
                     /note="incP origin of transfer"
ORIGIN
        1 cccggctgac gccgttggat accccaagga aagtctacac attcaggctg cgcaactgtt
       61 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
      121 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
      181 cggccagtga gcgcgcgtaa tacgactcac tatagggcga attgggtacc agttacgcta
      241 gggataacag ggtaatatag agagcgcttt tgaagctggg gtgggcgaag aactccagca
      301 tgagatcccc gcgctggagg atcatccagc cggcgtcccg gaaaacgatt ccgaagccca
      361 acctttcata gaaggcggcg gtggaatcga aatctcgtga tggcaggttg ggcgtcgctt
      421 ggtcggtcat ttcgaacccc agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa
      481 ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca
      541 ttcgccgcca agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc
      601 cgccacaccc agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat
      661 attcggcaag caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc
      721 cttgagcctg gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc
      781 ctgatcgaca agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg
      841 gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat
      901 gatggatact ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc
      961 gcccaatagc agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg
     1021 aacgcccgtc gtggccagcc acgatagccg cgctgcctcg tcctgcagtt cattcagggc
     1081 accggacagg tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac
     1141 ggcggcatca gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac
     1201 ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca
     1261 tcctgtctct tgatcagatc ttgatcccct gcgccatcag atccttggcg gcaagaaagc
     1321 catccagttt actttgcagg gcttcccaac cttaccagag ggcgccccag ctggcaattc
     1381 cggttcgctt gctgtccata aaaccgccca gtctagctat cgccatgtaa gcccactgca
     1441 agctacctgc tttctctttg cgcttgcgtt ttcccttgtc cagatagccc agtctcgagg
     1501 tcgacggtat cgataagctt gatatcgaat tcctgcagcc cgggggatcc actagttcta
     1561 gagcggccgc caccgcggtg gagctccagc ttttgttccc tttagtgagg gttaattgcg
     1621 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     1681 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     1741 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     1801 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     1861 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     1921 gctcactcaa aggcggtaat acggttatcc ccatgtcagc cgttaagtgt tcctgtgtca
     1981 ctcaaaattg ctttgagagg ctctaagggc ttctcagtgc gttacatccc tggcttgttg
     2041 tccacaaccg ttaaacctta aaagctttaa aagccttata tattcttttt tttcttataa
     2101 aacttaaaac cttagaggct atttaagttg ctgatttata ttaattttat tgttcaaaca
     2161 tgagagctta gtacgtgaaa catgagagct tagtacgtta gccatgagag cttagtacgt
     2221 tagccatgag ggtttagttc gttaaacatg agagcttagt acgttaaaca tgagagctta
     2281 gtacgtgaaa catgagagct tagtacgtac tatcaacagg ttgaactgct gatcttcaga
     2341 tcctctacgc cggacgcatc gtgggtggtt aactcgacat cttggttacc gtgaagttac
     2401 catcacggaa aaaggttatg ctgcttttaa gacccacttt cacatttaag ttgtttttct
     2461 aatccgcata tgatcaattc aaggccgaat aagaaggctg gctctgcacc ttggtgatca
     2521 aataattcga tagcttgtcg taataatggc ggcatactat cagtagtagg tgtttccctt
     2581 tcttctttag cgacttgatg ctcttgatct tccaatacgc aacctaaagt aaaatgcccc
     2641 acagcgctga gtgcatataa tgcattctct agtgaaaaac cttgttggca taaaaaggct
     2701 aattgatttt cgagagtttc atactgtttt tctgtaggcc gtgtacctaa atgtactttt
     2761 gctccatcgc gatgacttag taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa
     2821 tcttgccagc tttccccttc taaagggcaa aagtgagtat ggtgcctatc taacatctca
     2881 atggctaagg cgtcgagcaa agcccgctta ttttttacat gccaatacaa tgtaggctgc
     2941 tctacaccta gcctctgggc gagtttacgg gttgttaaac cttcgattcc gacctcatta
     3001 agcagctcta atgcgctgct aatcacttta cttttatcta atctagacat cattaattcc
     3061 taatttttgt tgacactcta tcattgatag agttatttta ccactcccta tcagtgatag
     3121 agaaaagtga aatgcatcaa aaaaaccagg taatgaacct gggtccgaac tctaaactgc
     3181 tgaaagaata caaatcccag ctgatcgaac tgaacatcga acagttcgaa gcaggtatcg
     3241 gtctgatcct gggtgatgct tacatccgtt ctcgtgatga aggtaaaacc tactgtatgc
     3301 agttcgagtg gaaaaacaaa gcatacatgg accacgtatg tctgctgtac gatcagtggg
     3361 tactgtcccc gccgcacaaa aaagaacgtg ttaaccacct gggtaacctg gtaatcacct
     3421 ggggcgccca gactttcaaa caccaagctt tcaacaaact ggctaacctg ttcatcgtta
     3481 acaacaaaaa aaccatcccg aacaacctgg ttgaaaacta cctgaccccg atgtctctgg
     3541 catactggtt catggatgat ggtggtaaat gggattacaa caaaaactct accaacaaat
     3601 cgatcgtact gaacacccag tctttcactt tcgaagaagt agaatacctg gttaagggtc
     3661 tgcgtaacaa attccaactg aactgttacg taaaaatcaa caaaaacaaa ccgatcatct
     3721 acatcgattc tatgtcttac ctgatcttct acaacctgat caaaccgtac ctgatcccgc
     3781 agatgatgta caaactgccg aacactatct cctccgaaac tttcctgaaa taataagtca
     3841 aggagacatg aacgatgaac atcaaaaagt ttgcaaaaca agcaacagta ttaaccttta
     3901 ctaccgcact gctggcagga ggcgcaactc aagcgtttgc gaaagaaacg aaccaaaagc
     3961 catataagga aacatacggc atttcccata ttacacgcca tgatatgctg caaatccctg
     4021 aacagcaaaa aaatgaaaaa tatcaagttc ctgagttcga ttcgtccaca attaaaaata
     4081 tctcttctgc aaaaggcctg gacgtttggg acagctggcc attacaaaac gctgacggca
     4141 ctgtcgcaaa ctatcacggc taccacatcg tctttgcatt agccggagat cctaaaaatg
     4201 cggatgacac atcgatttac atgttctatc aaaaagtcgg cgaaacttct attgacagct
     4261 ggaaaaacgc tggccgcgtc tttaaagaca gcgacaaatt cgatgcaaat gattctatcc
     4321 taaaagacca aacacaagaa tggtcaggtt cagccacatt tacatctgac ggaaaaatcc
     4381 gtttattcta cactgatttc tccggtaaac attacggcaa acaaacactg acaactgcac
     4441 aagttaacgt atcagcatca gacagctctt tgaacatcaa cggtgtagag gattataaat
     4501 caatctttga cggtgacgga aaaacgtatc aaaatgtaca gcagttcatc gatgaaggca
     4561 actacagctc aggcgacaac catacgctga gagatcctca ctacgtagaa gataaaggcc
     4621 acaaatactt agtatttgaa gcaaacactg gaactgaaga tggctaccaa ggcgaagaat
     4681 ctttatttaa caaagcatac tatggcaaaa gcacatcatt cttccgtcaa gaaagtcaaa
     4741 aacctctgca aagcgataaa aaacgcacgg ctgagttagc aaacggcgct ctcggtatga
     4801 ttgagctaaa cgatgattac acactgaaaa aagtgatgaa accgctgatt gcatctaaca
     4861 cagtaacaga tgaaattgaa cgcgcgaacg tctttaaaat gaacggcaaa tggtatctgt
     4921 tcactgactc ccgcggatca aaaatgacga ttgacggcat tacgtctaac gatatttaca
     4981 tgcttggtta tgtttctaat tctttaactg gcccatacaa gccgctgaac aaaactggcc
     5041 ttgtgttaaa aatggatctt gatcctaacg atgtaacctt tacttactca cacttcgctg
     5101 tacctcaagc gaaaggaaac aatgtcgtga ttacaagcta tatgacaaac agaggattct
     5161 acgcagacaa acaatcaacg tttgcgccta gcttcctgct gaacatcaaa ggcaagaaaa
     5221 catctgttgt caaagacagc atccttgaac aaggacaatt aacagttaac aaataaaatt
     5281 ggtcgacaag cttgatctgg cttatcgaaa ttaattagcg gctaaaaaag tcatggctct
     5341 gccctcgggc ggaccacgcc catcatgacc ttgccaagct cgtcctgctt ctcttcgatc
     5401 ttcgccagca gggcgaggat cgtggcatca ccgaaccgcg ccgtgcgcgg gtcgtcggtg
     5461 agccagagtt tcagcaggcc gcccaggcgg cccaggtcgc cattgatgcg ggccagctcg
     5521 cggacgtgct catagtccac gacgcccgtg attttgtagc cctggccgac ggccagcagg
     5581 taggccgaca ggctcatgcc ggccgccgcc gccttttcct caatcgctct tcgttcgtct
     5641 ggaaggcagt acaccttgat aggtgggctg cccttcctgg ttggcttggt ttcatcagcc
     5701 atccgcttgc cctcatctgt tacgccggcg gtagccggcc agcctcgcag agcaggattc
     5761 ccgttgagca ccgccaggtg cgaataaggg acagtgaaga aggaacaccc gctcgcgggt
     5821 gggcctactt cacctatcct g
//
