LOCUS       Exported                6083 bp ds-DNA     circular SYN 26-NOV-2018
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6083)
  AUTHORS   MIT
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Jan 20, 2020 from SnapGene 4.3.11
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6083
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          3134..4050
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     primer_bind     1..30
                     /label=mmD661
     misc_feature    1
                     /label=origin
     misc_recomb     118..2262
                     /label=NluI fragment from pKNOCK
                     /note="NluI fragment from pKNOCK"
     CDS             complement(156..890)
                     /label=ermG
                     /note="ermG"
     CDS             1378..2239
                     /label=beta-lactamase from pBR322
                     /note="beta-lactamase from pBR322"
     primer_bind     1837..1856
                     /label=oMT203
     primer_bind     complement(2089..2106)
                     /label=oMT1189
     primer_bind     2089..2106
                     /label=oMT1184
     misc_feature    2097
                     /label=t2096a
     misc_recomb     2277..3062
                     /label=RP4 oriT/RSK oriV
                     /note="RP4 oriT/RSK oriV"
     primer_bind     complement(2340..2359)
                     /label=oMT204
     primer_bind     2968..2987
                     /label=pNBU1_seq.FOR
     misc_feature    3062
                     /label=3062 mutation from ""g"" to ""t""
                     /note="Mutated in pMT1fix2, but will not fix, since it's at
                     the backbone of the vector and is not CDS or a region with 
                     important UTR."
     misc_feature    3063..3122
                     /label=pNBU2-Lac-dCas9-NL3 PCR Product - pNBU2-Lac-dCas9-
                     /note="Geneious type: Inserted sequence"
     terminator      3078..3107
                     /label=tL17 terminator
                     /label=tL17
     primer_bind     complement(3104..3134)
                     /label=oMT1185
     primer_bind     3123..3158
                     /label=oMT1338
     misc_feature    3130..3133
                     /label=CGCT
     misc_feature    3134..3416
                     /label=PAM3
     promoter        3134
                     /note="cfxA_promoter"
     promoter        3134
                     /label=PcfxA
                     /note="PcfxA promoter"
     promoter        3134
                     /note="lac UV5 promoter"
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    3377..3380
                     /label=-33
     primer_bind     complement(3395..3417)
                     /label=oMT1130
     misc_feature    3403..3410
                     /label=-7
     misc_feature    3416
                     /label=+1
                     /note="+1"
     misc_feature    3417..3491
                     /label=RiboJ00
                     /note="RiboJ00"
     primer_bind     3417..3434
                     /label=oMT1123
     primer_bind     3417..3434
                     /label=oMT1128
     RBS             3417
                     /label=BT1311_RBS
                     /note="BT1311_RBS"
     terminator      3492..3534
                     /note="rpiL*"
                     /note="rpiL* termninator"
     primer_bind     complement(3530..3559)
                     /label=oMT641
     CDS             3535..4050
                     /codon_start=1
                     /label=Luciferase Reporter
                     /note="Luciferase Reporter"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     primer_bind     3535..3559
                     /label=oMT41
     primer_bind     3823..3842
                     /label=oMT399
     primer_bind     complement(4029..4055)
                     /label=oMT1339
     CDS             4048..4050
                     /codon_start=1
                     /label=CmeR(NCTC11168)
                     /note="CmeR(NCTC11168)"
                     /translation=""
     gene            4048..4050
                     /gene="rpoD"
                     /gene_synonym="alt; ECK3057; JW3039"
                     /locus_tag="b3067"
                     /label=rpoD
                     /note="rpoD"
                     /db_xref="EcoGene:EG10896"
                     /db_xref="GeneID:947567"
     CDS             4048..4050
                     /codon_start=1
                     /label=BreR-Bt
                     /note="BreR-Bt"
                     /translation=""
     primer_bind     4051..4075
                     /label=oMT1188
     misc_feature    4051..4060
                     /label=Amp up this region
     misc_feature    4051..4054
                     /label=CAAT
     primer_bind     4061..4084
                     /label=oMT367
     misc_feature    4061..4066
                     /label=problematic assembly region
     terminator      4067..4127
                     /label=L3S2P21 Terminator
     primer_bind     complement(4106..4146)
                     /label=oMT1272
     misc_feature    4106..4127
                     /label=Feature 67
     primer_bind     4128..4155
                     /label=oMT1259
     primer_bind     complement(4253..4273)
                     /label=pNBU1_seq.REV
     misc_feature    complement(4302..4340)
                     /label=mmD660
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    4341..6083
                     /label=intN1 Construct PCR Product - intN1 Construct P
                     /label=intN1 Construct PCR Product - intN1 Construct PCR 
                     Product 2
                     /note="Geneious type: Inserted sequence"
     misc_feature    4341..5140
                     /label=Piece1
     misc_feature    4341..4370
                     /label=Geneious type: Editing History Insertion
                     /note="Geneious type: Editing History Insertion"
     primer_bind     4341..4369
                     /label=mmD658
     primer_bind     4362..4382
                     /label=oMT113
     misc_feature    4371..4700
                     /label=attN1 Region
     primer_bind     4371..4390
                     /label=intN1 Construct PCR Product (fwd) - pNBU2-PcfxA
                     /label=intN1 Construct PCR Product (fwd) - 
                     pNBU2-PcfxA-NanoLuc PCR Product
     misc_feature    4421..4436
                     /label=DR1
     misc_feature    4454..4468
                     /label=IR1
     misc_feature    4478..4492
                     /label=IR2
     misc_feature    4498..4511
                     /label=CC Region
     misc_feature    4510
                     /label=G-->C
     misc_feature    4510
                     /label=G
                     /note="Geneious type: Editing History Replacement"
     misc_feature    4511
                     /label='-1'
     misc_feature    4512
                     /label='+1'
     misc_feature    4529..4544
                     /label=DR2
     primer_bind     complement(4567..4589)
                     /label=oMT114
     misc_feature    4653..4669
                     /label=DR3
     CDS             4716..5140
                     /codon_start=1
                     /transl_table=11
                     /gene="intN1"
                     /product="IntN1"
                     /label=intN1
                     /label=intN1 CDS
                     /note="NBU1 integrase"
                     /db_xref="GI:8308020"
                     /protein_id="AAF74437.1"
                     /translation="MKVTFIIKKAAKRYDTESMATIYVRFRNGRQLDSVAPTQLAINPN
                     LWDDKDECVKTKAVCNEEMRTHINEEIRQLKTYIEKVYQQEKEAIDKEWLKTTLDKFYH
                     PEKYFLPEEVVIKPTIGELFDEFLNKHPLSEVRKKNF"
     gene            4716..5140
                     /gene="intN1"
                     /label=intN1
                     /label=intN1 gene
     primer_bind     complement(4932..4959)
                     /label=oMT157
     primer_bind     4932..4959
                     /label=oMT156
     misc_feature    complement(5110..5140)
                     /label=mmD659
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    5111..6083
                     /label=Piece2
     CDS             5111..6053
                     /codon_start=1
                     /transl_table=11
                     /gene="intN1"
                     /product="IntN1"
                     /label=intN1
                     /label=intN1 CDS
                     /note="NBU1 integrase"
                     /db_xref="GI:8308020"
                     /protein_id="AAF74437.1"
                     /translation="MVGSTKEKFPGCQKSLTAL*TICKGYKERTKGLYP*CGFGNT*HA
                     SGYVGFLSERIPVL*TLPEHL*SHSRKEDTTAQKQKHADRLFFKNTHILPVVLR*QTHH
                     KQTFRQVSDRGVYIWYTLLYNTRRKGQDF*CRPFCHPTTGNTEGYIHISDTDRMQGERP
                     VPNDQTKCGQ*SHRIYSQENQRGESGYGTCST*RQSERNP*TLQRI*GKTVAVHIRAKV
                     Q*CHKKDIQISWS*PHRNNLRPVDAQRNQTTYL*SGKQPSGKTYVYRQYL*KSERPEPC
                     FRTVGTQGGKQSFQTIQGY*RRNEERPCKTTGL"
     gene            5111..6053
                     /gene="intN1"
                     /label=intN1
                     /label=intN1 gene
     misc_feature    5111..5140
                     /label=Piece2
     misc_feature    5111..5140
                     /label=Ligation
     primer_bind     5111..5130
                     /label=intN1 Construct PCR Product 2 (fwd)
     misc_feature    complement(5116..5140)
                     /label=intN1 Construct PCR Product (rev)
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(6029..6055)
                     /label=intN1 Construct PCR Product 2 (rev) - pNBU2-Pcf
                     /label=intN1 Construct PCR Product 2 (rev) - 
                     pNBU2-PcfxA-NanoLuc PCR Product
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    6045
                     /label=T
                     /note="Geneious type: Editing History Replacement"
     misc_feature    complement(6047..6083)
                     /label=mmD661
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    6047
                     /label=A
                     /note="Geneious type: Editing History Replacement"
     misc_feature    6054..6083
                     /label=HOMOLOGY
     misc_feature    6056..6083
                     /label=CTTGTAGTTCGTTTAATTCGCTTTTCGTTTTTACGAATTTAACGAAT
                     /label=CTTGTAGTTCGTTTAATTCGCTTTTCGTTTTTACGAATTTAACGAATTGA 
                     ATATCATTGTTTTACGATTGATTTTCATTATATATTTGCGCCACTATCCTAATCGGGG 
                     TAGTGGCTTTTCGTTTATAATACTGCCGAAACGGGAA
                     /note="Geneious type: Editing History Replacement"
ORIGIN
        1 agagtagcga tattaaaaga atccgatgag aaaagactaa tatttatcta tccattcagt
       61 ttgatttttc aggactttac atcgtcctga aagtatttgt tggtaccggt accgaggacg
      121 cgtaaacatt tacagttgca tgtggcctat tgtttttagc cgttaaatat tttataacta
      181 ttaaatagcg atacaaattg ttcgaaacta atattgttta tatcatatat tctcgcatgt
      241 tttaaagctt tattaaattg attttttgta aacagttttt cgtactcttt gttaacccat
      301 ttcattacaa aagtttcata tttttttctc tctttaaatg ccatttttgc tggctttctt
      361 tttaatacaa ttaatgtgct atccacttta ggttttggat ggaaataata cctaggaatt
      421 tttgctaata tagaaatatc tacctctgcc attaacagca atgctagtga tctgtttgta
      481 tctaataaca ttttagcaaa accatattcc actattaaat aacttattgt ggctgaactt
      541 tcaaaaacaa tttttcgaat tatatttgtg cttatgttgt aaggtatgct gccaaatatt
      601 ttatatggat tgtggctagg aaatgtaaat ttcagtatat catcatttac tatttgatag
      661 ttaggataat ttaagagctt attacgagtt acctcacata atttagaatc aatttctatc
      721 gccgttacaa aattacatct ctttaccaat ccagcagtaa aatgaccttt ccctgcacct
      781 atttcaaaga tgttatcttt ttcatctaaa cttatgcaat tcattatttt ttctatgtga
      841 tattttgaag taataaaatt ttgactatct tttatattta ctttgttcat tataacctct
      901 ccttaattta ttgcatctct tttcgaatat ttatgttttt tgagaaaaga acgtactcat
      961 ggttcatccc gatatgcgta tcggtctgta tatcagcaac tttctatgtg tttcaactac
     1021 aatagtcatc tattctcatc tttctgagtc caccccctgc aaagcccctc tttacgacat
     1081 aaaaattcgg tcggaaaagg tatgcaaaag atgtttctct ctttaagaga aactcttcgg
     1141 gatgcaaaaa tatgaaaata actccaattc accaaattat atagcgactt ttttacaaaa
     1201 tgctaaaatt tgttgatttc cgtcaagcaa ttgttgagca aaaatgtctt ttacgataaa
     1261 atgatacctc aatatcaact gtttagcaaa acgatatttc tcttaaagag agaaacacct
     1321 ttttgttcac caatccccga cttttaatcc cgcggccatg attgaaaaag gaagagtatg
     1381 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     1441 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     1501 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     1561 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     1621 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     1681 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     1741 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     1801 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     1861 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     1921 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     1981 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     2041 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtcacgc
     2101 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     2161 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     2221 ctgattaagc attggtaact gtcagaccaa gtttactcat aacgcgtcaa ttcgaggggg
     2281 atcaattccg tgataggtgg gctgcccttc ctggttggct tggtttcatc agccatccgc
     2341 ttgccctcat ctgttacgcc ggcggtagcc ggccagcctc gcagagcagg attcccgttg
     2401 agcaccgcca ggtgcgaata agggacagtg aagaaggaac acccgctcgc gggtgggcct
     2461 acttcaccta tcctgcccgg ctgacgccgt tggatacacc aaggaaagtc tacacgaacc
     2521 ctttggcaaa atcctgtata tcgtgcgaaa aaggatggat ataccgaaaa aatcgctata
     2581 atgaccccga agcagggtta tgcagcggaa aacggaattg atccggccac gatgcgtccg
     2641 gcgtagagga tctgaagatc agcagttcaa cctgttgata gtacgtacta agctctcatg
     2701 tttcacgtac taagctctca tgtttaacgt actaagctct catgtttaac gaactaaacc
     2761 ctcatggcta acgtactaag ctctcatggc taacgtacta agctctcatg tttcacgtac
     2821 taagctctca tgtttgaaca ataaaattaa tataaatcag caacttaaat agcctctaag
     2881 gttttaagtt ttataagaaa aaaaagaata tataaggctt ttaaagcttt taaggtttaa
     2941 cggttgtgga caacaagcca gggatgtaac gcactgagaa gcccttagag cctctcaaag
     3001 caattttgag tgacacagga acacttaacg gctgacatgg gaattcccct ccaccgcggt
     3061 ggattaatgc ggctgcaatt tttttgggcg gggccgccca aaaaaatcct agcaccctgc
     3121 agggtctccc gcttgatctg gaagaagcaa tgaaagctgc tgttaagtct ccgaatcagg
     3181 tattgttcct gacaggtgta ttcccatccg gtaaacgcgg atactttgca gttgatctga
     3241 ctcaggaata aattataaat taaggtaaga agattgtagg ataagctaat gaaatagaaa
     3301 aaggatgccg tcacacaact tgtcggcatt cttttttgtt ttattagttg aaaatatagt
     3361 gaaaactttg caacagcata gctcagcaca gattatttgt cgtaactttg cactccagct
     3421 gtcaccggat gtgctttccg gtctgatgag tccgtgagga cgaaacagcc tctacaaata
     3481 attttgttta acgcatttta aaataaaata aattatttat ttaattaaac gaatatggtt
     3541 tttactctgg aagattttgt tggcgattgg cgtcagaccg cgggttataa tttggatcaa
     3601 gtcctggaac agggtggcgt aagctctctg ttccagaacc tgggtgtgag cgtgacgccg
     3661 attcagcgca tcgttctgtc cggcgagaac ggtctgaaaa ttgatattca tgtgatcatc
     3721 ccgtacgaag gcctgagcgg tgaccaaatg ggtcaaatcg agaaaatctt taaagtcgtc
     3781 tacccagttg acgatcacca cttcaaggtt atcttgcatt acggtacgct ggtgattgat
     3841 ggtgtgaccc cgaatatgat tgactatttc ggccgtccgt atgaaggcat tgccgttttt
     3901 gacggtaaaa agatcaccgt caccggtacc ctgtggaatg gcaataagat tattgacgag
     3961 cgtctgatta acccggacgg cagcctgctg ttccgcgtga ccatcaacgg tgtcacgggt
     4021 tggcgtctgt gcgagcgcat cctggcataa caatgaatgg gctttcctcg gtaccaaatt
     4081 ccagaaaaga ggcctcccga aaggggggcc ttttttcgtt ttggtcccta attgcctatc
     4141 ttccagtgat ggaacagcat ttgtgcattg gctgcaacaa tcagccttac ttgtgcctgt
     4201 tctatttccg aaccgaccgc ttgtatgaat ccatcaaaat tcgttttctc tatgttggat
     4261 tccttgttgc tcatattgtg atgataattt ctacaaatat agtcattggt aactatctat
     4321 gaaactgttt gatactttta ctcatcggat tcttttaata tcgctactct ctacgttcaa
     4381 ccaaaagaaa taatgactta ctgctatatt ttttgcacgt gtggggaaaa tgtggggaaa
     4441 attcaagcaa aagaaaaagc taagtattga actatcaaat acttagcttt ctttcttgta
     4501 cccagacccc gcatttgaaa taattaaagt ggggaaaatg tgggtaaaaa gaaaaatgcg
     4561 gaaaaacgcc acaattacac tgtatttcaa tatgttataa tcctattaaa ttttaatcca
     4621 agtttaatcg aattgcaaaa tatttagcag atgtggggaa aatgctgggg aaaatattta
     4681 tatttgcagc agagtaaaat tcaacctgaa agtctatgaa agtaaccttt atcattaaaa
     4741 aagcagccaa acgatatgat acagaatcca tggctacaat ctatgtccgt tttagaaacg
     4801 gaaggcagtt agactccgtt gctcctactc agttagccat caatcccaat ctatgggatg
     4861 ataaagacga atgtgtaaaa acgaaagctg tctgcaatga agaaatgcgt acccatataa
     4921 atgaagagat acgccagttg aaaacctata tcgagaaggt atatcaacaa gaaaaggaag
     4981 caatagacaa agaatggcta aaaacaacac ttgataaatt ttaccatcct gaaaaatatt
     5041 ttttgccgga ggaagtggtt atcaagccta ccattggaga actattcgat gaatttctaa
     5101 acaagcaccc tttgtcggaa gtacgaaaga aaaatttccg ggttgtcaaa agagccttac
     5161 tgcgttatga actatatgta agggctacaa agagaggaca aaagggcttt atccttgatg
     5221 tggatttggt aacacctgac acgcttcggg atatgtggga tttctttcag aacgaatacc
     5281 agtattatga actttacccg agcatttatg aagccattcc cgaaaagagg acaccacagc
     5341 ccagaagcaa aaacacgctg atagactgtt tttcaagaat acgcacattc ttcctgtggt
     5401 gcttcgataa caaacgcacc acaaacagac ctttcgacaa gtttccgata gaggagtgta
     5461 catatggtac accttattat ataacactcg aagaaaggga caggattttt aatgcagacc
     5521 tttctgccac cccacaactg gcaatacaga gggatatatt catatttcag acactgatag
     5581 gatgcagggt gagcgacctg taccgaatga ccaaactaaa tgtggtcaat gaagccatag
     5641 aatatattcc caagaaaacc aaagagggga atccggttac ggtacgtgtt ccacttaacg
     5701 acaaagcgaa agaaatcctt gaacgctaca aagaatatga gggaaaactg ttgccgttca
     5761 tatccgagca aaagtacaat gatgccataa aaaagatatt caaattagct ggagttgacc
     5821 gcatcgtaac aatcttagac ccgttgacgc acaacgaaat caaacgacct atttatgaag
     5881 tggcaagcag ccatctggca agacgtacgt ttatcggcaa tatctataaa aaagtgaaag
     5941 acccgaacct tgtttccgca ctgtcgggac acaaggaggg aagcaaagct ttcagacgat
     6001 acagggatat tgacgaagaa atgaagaaag accttgtaaa actactggac tgataaaagt
     6061 atcaaacagt ttcatagata gtt
//
