LOCUS       Exported                7301 bp ds-DNA     circular SYN 14-FEB-2018
DEFINITION  synthetic circular DNA
ACCESSION   urn.local...1z-55o2kyd
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7301)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Feb 14, 2018 from SnapGene 4.1.4
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7301
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          1..1760
                     /note="/dnas_title=pAB108e - AP RepA SC101 gRNABS_mod 
                     pLac61*(434-OR1-m13_16) SD8 luxAB Plac G7'-gRNA(SP) bla"
     source          1761..7301
                     /note="/dnas_title=colEI plasmid"
     source          1761..1915
                     /note="/dnas_title=pAB045c - CP ColEI Rop pTet SD8 
                     rpoZ-cMyc-HA4 SpectR lacI"
     source          6038..6331
                     /note="/dnas_title=pAB108e - AP RepA SC101 gRNABS_mod 
                     pLac61*(434-OR1-m13_16) SD8 luxAB Plac G7'-gRNA(SP) bla"
     CDS             complement(1..951)
                     /label=RepA
                     /note="RepA"
                     /note="pSC101 origin of replication protein"
     primer_bind     300..315
                     /label=WT071 seq RepA 300 FP
     primer_bind     928..951
                     /label=WT072 seq
     rep_origin      complement(955..1700)
                     /direction=LEFT
                     /label=pSC101
                     /note="pSC101"
     primer_bind     1682..1700
                     /label=WT014 seq
     modified_base   1701..1715
                     /label=USER junction
                     /note="USER junction"
     terminator      1716..1760
                     /note="rrnB1 transcriptional terminator"
     primer_bind     complement(1739..1776)
                     /label=WT079
     misc_RNA        1753..1760
                     /label=gRNA binding site, discarded
                     /note="gRNA binding site"
                     /note="/modified_by=User"
     primer_bind     1761..1797
                     /label=WT005
     modified_base   1761..1776
                     /label=USER Linker
                     /note="USER Linker"
     protein_bind    1796..1814
                     /note="tetR binding site"
     promoter        1815..1850
                     /label=tet-regulated promoter
                     /note="anhydrotetracycline induction"
                     /note="/dnas_title=tet-regulated promoter"
                     /note="/vntifkey=30"
     protein_bind    1850..1868
                     /note="tetR binding site"
     primer_bind     complement(1898..1915)
                     /label=WT032 tet ColEI aadA RP from pWT002
     modified_base   1901..1915
                     /label=USER junction
                     /note="USER junction"
     primer_bind     1904..1951
                     /label=WT082
     RBS             1916..1930
                     /label=SD8
                     /note="SD8"
                     /note="Ringquist and Gold"
     misc_feature    1931..1933
                     /label=Start
                     /note="/modified_by=User"
     misc_feature    1934..6034
                     /label=Church Lab Cas9-m4
                     /note="/ApEinfo_fwdcolor=#0000ff"
                     /note="/ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {}
                     0} width 5 offset 0"
                     /note="/ApEinfo_revcolor=green"
                     /note="/modified_by=User"
     variation       1958..1960
                     /label=D10A
                     /label=nonstandard type: Mutation
                     /note="/created_by=User"
     primer_bind     complement(1999..2016)
                     /label=WT174 seq
     primer_bind     2410..2430
                     /label=WT061 seq
     primer_bind     complement(2843..2860)
                     /label=mCas9-3
     primer_bind     3010..3027
                     /label=WT062 seq
     primer_bind     3610..3629
                     /label=WT063 seq
     primer_bind     4210..4227
                     /label=WT064
     variation       4448..4450
                     /label=H840A
                     /label=nonstandard type: Mutation
                     /note="/created_by=User"
     primer_bind     4810..4830
                     /label=WT065 seq
     primer_bind     5410..5427
                     /label=WT066 seq
     primer_bind     5932..5949
                     /label=WT039 Cas9 4001-4018 FP
     primer_bind     complement(6020..6050)
                     /label=WT033 Cas9 RP from pJH573
     misc_feature    6035..6037
                     /label=Stop Codon
                     /note="/created_by=User"
     primer_bind     6038..6054
                     /label=WT034 RP sgRNA promoter from pWT003a
     misc_feature    6038..6050
                     /label=USER linker
                     /note="USER linker"
     terminator      6052..6084
                     /note="BBa_B1002"
     terminator      complement(6086..6124)
                     /note="BBa_B1006"
     modified_base   complement(6125..6143)
                     /label=USER Junction
                     /note="USER Junction"
     misc_feature    6144..6226
                     /label=gRNA backbone
                     /note="/dnas_title=gRNA backbone"
                     /note="/vntifkey=21"
     primer_bind     complement(6200..6258)
                     /label=WT246
     misc_feature    complement(6227..6246)
                     /label=173 spacer
                     /note="/ApEinfo_fwdcolor=#cb8b34"
                     /note="/ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {}
                     0} width 5 offset 0"
                     /note="/ApEinfo_revcolor=green"
                     /note="/modified_by=User"
     misc_feature    complement(6248..6286)
                     /label=lac promoter (no LacO)
                     /note="Geneious type: promoter prokaryotic"
     primer_bind     6250..6271
                     /label=WT239
     -10_signal      complement(6254..6259)
     misc_feature    6262..6273
                     /label=User juction
                     /label=nonstandard type: overhang
                     /note="/created_by=User"
     -35_signal      complement(6278..6283)
     protein_bind    complement(6302..6316)
                     /label=CAP binding site
     primer_bind     complement(6311..6348)
                     /label=WT035, sgRNA promoter FP from pWT003a
     primer_bind     6332..6355
                     /label=WT036, aadA ColEI from pWT002
     misc_feature    6332..6348
                     /label=USER linker
     terminator      complement(6356..6397)
                     /label=tetA/orfL bidirectional terminator
     modified_base   6398..6410
                     /label=USER junction
                     /note="USER junction"
     CDS             complement(6411..7202)
                     /codon_start=1
                     /label=aadA, streptomycin adenylyltransferase
                     /note="/dnas_title=aadA, streptomycin adenylyltransferase"
                     /note="/modified_by=User"
                     /note="/vntifkey=4"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     primer_bind     complement(6414..6433)
                     /label=WT040 aadA end FP sequencing
     primer_bind     6667..6676
                     /label=WT071 seq RepA 300 FP
     primer_bind     7185..7202
                     /label=WT050 seq
     promoter        7203..7287
                     /note="aadA promoter"
     primer_bind     complement(7267..7301)
                     /label=WT108 revised WT081
     primer_bind     7288..23
                     /label=WT080
     modified_base   7288..7301
                     /label=USER junction
                     /note="USER junction"
ORIGIN
        1 tcagatcctt ccgtatttag ccagtatgtt ctctagtgtg gttcgttgtt tttgcgtgag
       61 ccatgagaac gaaccattga gatcatgctt actttgcatg tcactcaaaa attttgcctc
      121 aaaactggtg agctgaattt ttgcagttaa agcatcgtgt agtgtttttc ttagtccgtt
      181 acgtaggtag gaatctgatg taatggttgt tggtattttg tcaccattca tttttatctg
      241 gttgttctca agttcggtta cgagatccat ttgtctatct agttcaactt ggaaaatcaa
      301 cgtatcagtc gggcggcctc gcttatcaac caccaatttc atattgctgt aagtgtttaa
      361 atctttactt attggtttca aaacccattg gttaagcctt ttaaactcat ggtagttatt
      421 ttcaagcatt aacatgaact taaattcatc aaggctaatc tctatatttg ccttgtgagt
      481 tttcttttgt gttagttctt ttaataacca ctcataaatc ctcatagagt atttgttttc
      541 aaaagactta acatgttcca gattatattt tatgaatttt tttaactgga aaagataagg
      601 caatatctct tcactaaaaa ctaattctaa tttttcgctt gagaacttgg catagtttgt
      661 ccactggaaa atctcaaagc ctttaaccaa aggattcctg atttccacag ttctcgtcat
      721 cagctctctg gttgctttag ctaatacacc ataagcattt tccctactga tgttcatcat
      781 ctgagcgtat tggttataag tgaacgatac cgtccgttct ttccttgtag ggttttcaat
      841 cgtggggttg agtagtgcca cacagcataa aattagcttg gtttcatgct ccgttaagtc
      901 atagcgacta atcgctagtt catttgcttt gaaaacaact aattcagaca tacatctcaa
      961 ttggtctagg tgattttaat cactatacca attgagatgg gctagtcaat gataattact
     1021 agtccttttc ctttgagttg tgggtatctg taaattctgc tagacctttg ctggaaaact
     1081 tgtaaattct gctagaccct ctgtaaattc cgctagacct ttgtgtgttt tttttgttta
     1141 tattcaagtg gttataattt atagaataaa gaaagaataa aaaaagataa aaagaataga
     1201 tcccagccct gtgtataact cactacttta gtcagttccg cagtattaca aaaggatgtc
     1261 gcaaacgctg tttgctcctc tacaaaacag accttaaaac cctaaaggct taagtagcac
     1321 cctcgcaagc tcgggcaaat cgctgaatat tccttttgtc tccgaccatc aggcacctga
     1381 gtcgctgtct ttttcgtgac attcagttcg ctgcgctcac ggctctggca gtgaatgggg
     1441 gtaaatggca ctacaggcgc cttttatgga ttcatgcaag gaaactaccc ataatacaag
     1501 aaaagcccgt cacgggcttc tcagggcgtt ttatggcggg tctgctatgt ggtgctatct
     1561 gactttttgc tgttcagcag ttcctgccct ctgattttcc agtctgacca cttcggatta
     1621 tcccgtgaca ggtcattcag actggctaat gcacccagta aggcagcggt atcatcaaca
     1681 ggcttacccg tcttactgtc aagaggacat ccggtcaaat aaaacgaaag gctcagtcga
     1741 aagactgggc ctttcgtttt attctagagc acagctccat cgaatggcca gatgatccct
     1801 atcagtgata gagattgaca ctctatcatt gatagagtta ttttaccact ccctatcagt
     1861 gatagagaaa agtgaaatga atagttcgac aaaaatctag acctgcaggt gcagtaagga
     1921 ggaaaaaaaa atggacaaga agtactccat tgggctcgat atcggcacaa acagcgtcgg
     1981 ctgggccgtc attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa
     2041 taccgatcgc cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga
     2101 gacggccgaa gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa
     2161 tcggatctgc tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt
     2221 cttccatagg ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc
     2281 aatctttggc aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca
     2341 tctgaggaag aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc
     2401 gctggcgcat atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga
     2461 caacagcgat gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga
     2521 agagaacccg atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc
     2581 caaatcccgg cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct
     2641 gtttggtaat cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga
     2701 cctggccgaa gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa
     2761 tctgctggcc cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc
     2821 agacgccatt ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct
     2881 gagcgctagt atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc
     2941 ccttgtcaga cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa
     3001 tggctacgcc ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa
     3061 gcccatcttg gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga
     3121 tctgttgcgc aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg
     3181 cgaactgcac gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag
     3241 ggaaaagatt gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg
     3301 gggaaattcc agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa
     3361 cttcgaggaa gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa
     3421 ctttgataaa aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta
     3481 cttcacagtt tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc
     3541 agcattcctg tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg
     3601 gaaagttacc gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc
     3661 tgttgaaatc agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct
     3721 cctgaaaatc attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga
     3781 ggacattgtc ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa
     3841 aacttacgct catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac
     3901 aggatggggg cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa
     3961 gacaatcctg gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat
     4021 ccatgatgac tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg
     4081 ggacagtctt cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat
     4141 actgcagacc gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga
     4201 gaatatcgtt atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag
     4261 tagggaaagg atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa
     4321 ggaacaccca gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca
     4381 gaacggcagg gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga
     4441 cgtggaccat atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt
     4501 gacaagatcc gataaaaaca gagggaagag tgataacgtc ccctcagaag aagttgtcaa
     4561 gaaaatgaaa aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt
     4621 cgataatctg actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat
     4681 caaaaggcag cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc
     4741 acgcatgaac accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac
     4801 tctgaagtct aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga
     4861 gatcaacaat taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact
     4921 tatcaaaaaa tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga
     4981 tgttaggaaa atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt
     5041 cttttacagc aatattatga attttttcaa gaccgagatt acactggcca atggagagat
     5101 tcggaagcga ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg
     5161 tagggatttc gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa
     5221 gaccgaagta cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga
     5281 caagctgatc gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc
     5341 tacagtcgct tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact
     5401 caaaagcgtc aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa
     5461 ccccatcgac tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa
     5521 gcttcccaag tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc
     5581 gggcgagctg cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta
     5641 tctggccagc cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct
     5701 gttcgtggaa caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc
     5761 caaaagagtg atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca
     5821 cagggataag cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa
     5881 cttgggcgcg cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac
     5941 ctctacaaag gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga
     6001 aacaagaatc gacctctctc agctcggtgg agactgaacg gagccaatgt acgcaaaaac
     6061 cccgcttcag cggggttttt tcgccaaaaa aaaccccgcc ctgtcagggg cggggttttt
     6121 ttttatgttg aaaatctcct tctaaaaaaa gcaccgactc ggtgccactt tttcaagttg
     6181 ataacggact agccttattt taacttgcta tttctagctc taaaaccact gtggggtgga
     6241 ggggactcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     6301 agtgagctaa ctcacattaa ttgcgttgcg cacttaatta acggcactcc tcagcaaata
     6361 taatgaccct cttgataacc caagagggca ttttttaatg cccatggcgt ttatttgccg
     6421 actaccttgg tgatctcgcc tttcacgtag tggacaaatt cttccaactg atctgcgcgc
     6481 gaggccaagc gatcttcttc ttgtccaaga taagcctgtc tagcttcaag tatgacgggc
     6541 tgatactggg ccggcaggcg ctccattgcc cagtcggcag cgacatcctt cggcgcgatt
     6601 ttgccggtta ctgcgctgta ccaaatgcgg gacaacgtaa gcactacatt tcgctcatcg
     6661 ccagcccagt cgggcggcga gttccatagc gttaaggttt catttagcgc ctcaaataga
     6721 tcctgttcag gaaccggatc aaagagttcc tccgccgctg gacctaccaa ggcaacgcta
     6781 tgttctcttg cttttgtcag caagatagcc agatcaatgt cgatcgtggc tggctcgaag
     6841 atacctgcaa gaatgtcatt gcgctgccat tctccaaatt gcagttcgcg cttagctgga
     6901 taacgccacg gaatgatgtc gtcgtgcaca acaatggtga cttctacagc gcggagaatc
     6961 tcgctctctc caggggaagc cgaagtttcc aaaaggtcgt tgatcaaagc tcgccgcgtt
     7021 gtttcatcaa gccttacggt caccgtaacc agcaaatcaa tatcactgtg tggcttcagg
     7081 ccgccatcca ctgcggagcc gtacaaatgt acggccagca acgtcggttc gagatggcgc
     7141 tcgatgacgc caactacctc tgatagttga gtcgatactt cggcgatcac cgcttccctc
     7201 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     7261 tacatatttg aatgtattta gaaaaatagg ccaaatgccg t
//
