LOCUS       Exported                5943 bp ds-DNA     circular SYN 20-JUN-2018
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5943)
  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..5943
                     /organism="synthetic DNA construct"
                     /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(3099..3122)
                     /label=oMT721
     primer_bind     complement(3104..3134)
                     /label=oMT1185
     primer_bind     3123..3170
                     /label=oMT1340
     misc_feature    3123..3130
                     /label=L3S3P21
     misc_feature    3130..3133
                     /label=CGCT
     promoter        3134..3276
                     /label=IPTG Promoter
     protein_bind    3183..3199
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     protein_bind    3216..3232
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     complement(3242..3281)
                     /label=oMT715
     primer_bind     3276..3294
                     /label=oMT716
     misc_feature    3277..3351
                     /label=RiboJ00
     misc_feature    3277..3280
                     /label=4bp-2
     terminator      3352..3394
                     /label=rpiL*
                     /note="rpiL* termninator"
     primer_bind     complement(3390..3419)
                     /label=oMT641
     CDS             3395..3910
                     /codon_start=1
                     /label=Luciferase Reporter
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     primer_bind     3395..3419
                     /label=oMT41
     primer_bind     3683..3702
                     /label=oMT399
     primer_bind     complement(3889..3915)
                     /label=oMT1339
     misc_feature    3911..3914
                     /label=CAAT
     misc_feature    3921..3926
                     /label=problematic assembly region
     terminator      3927..3987
                     /label=L3S2P21 Terminator
     primer_bind     complement(3966..4006)
                     /label=oMT1272
     primer_bind     3988..4015
                     /label=oMT1259
     primer_bind     complement(4113..4133)
                     /label=pNBU1_seq.REV
     misc_feature    complement(4162..4200)
                     /label=mmD660
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    4201..5943
                     /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    4201..5000
                     /label=Piece1
     misc_feature    4201..4230
                     /label=Geneious type: Editing History Insertion
                     /note="Geneious type: Editing History Insertion"
     primer_bind     4201..4229
                     /label=mmD658
     primer_bind     4222..4242
                     /label=oMT113
     misc_feature    4231..4560
                     /label=attN1 Region
     primer_bind     4231..4250
                     /label=intN1 Construct PCR Product (fwd) - pNBU2-PcfxA
                     /label=intN1 Construct PCR Product (fwd) - 
                     pNBU2-PcfxA-NanoLuc PCR Product
     misc_feature    4281..4296
                     /label=DR1
     misc_feature    4314..4328
                     /label=IR1
     misc_feature    4338..4352
                     /label=IR2
     misc_feature    4358..4371
                     /label=CC Region
     misc_feature    4370
                     /label=G
                     /note="Geneious type: Editing History Replacement"
     misc_feature    4370
                     /label=G-->C
     misc_feature    4371
                     /label='-1'
     misc_feature    4372
                     /label='+1'
     misc_feature    4389..4404
                     /label=DR2
     primer_bind     complement(4427..4449)
                     /label=oMT114
     misc_feature    4513..4529
                     /label=DR3
     gene            4576..5000
                     /gene="intN1"
                     /label=intN1
                     /label=intN1 gene
     CDS             4576..5000
                     /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"
     primer_bind     complement(4792..4819)
                     /label=oMT157
     primer_bind     4792..4819
                     /label=oMT156
     misc_feature    complement(4970..5000)
                     /label=mmD659
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    4971..5943
                     /label=Piece2
     gene            4971..5913
                     /gene="intN1"
                     /label=intN1
                     /label=intN1 gene
     CDS             4971..5913
                     /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"
     misc_feature    4971..5000
                     /label=Ligation
     misc_feature    4971..5000
                     /label=Piece2
     primer_bind     4971..4990
                     /label=intN1 Construct PCR Product 2 (fwd)
     misc_feature    complement(4976..5000)
                     /label=intN1 Construct PCR Product (rev)
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(5889..5915)
                     /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    5905
                     /label=T
                     /note="Geneious type: Editing History Replacement"
     misc_feature    complement(5907..5943)
                     /label=mmD661
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    5907
                     /label=A
                     /note="Geneious type: Editing History Replacement"
     misc_feature    5914..5943
                     /label=HOMOLOGY
     misc_feature    5916..5943
                     /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 gctttacaaa gaaaattcga caaactgtta tttttctatc tatttatttg
     3181 aattgtgagc ggataacaat tacctttgtc ggcaattgtg agcggataac aattaaataa
     3241 agatattctc gtcaaacaaa tataaataat ataaacagct gtcaccggat gtgctttccg
     3301 gtctgatgag tccgtgagga cgaaacagcc tctacaaata attttgttta acgcatttta
     3361 aaataaaata aattatttat ttaattaaac gaatatggtt tttactctgg aagattttgt
     3421 tggcgattgg cgtcagaccg cgggttataa tttggatcaa gtcctggaac agggtggcgt
     3481 aagctctctg ttccagaacc tgggtgtgag cgtgacgccg attcagcgca tcgttctgtc
     3541 cggcgagaac ggtctgaaaa ttgatattca tgtgatcatc ccgtacgaag gcctgagcgg
     3601 tgaccaaatg ggtcaaatcg agaaaatctt taaagtcgtc tacccagttg acgatcacca
     3661 cttcaaggtt atcttgcatt acggtacgct ggtgattgat ggtgtgaccc cgaatatgat
     3721 tgactatttc ggccgtccgt atgaaggcat tgccgttttt gacggtaaaa agatcaccgt
     3781 caccggtacc ctgtggaatg gcaataagat tattgacgag cgtctgatta acccggacgg
     3841 cagcctgctg ttccgcgtga ccatcaacgg tgtcacgggt tggcgtctgt gcgagcgcat
     3901 cctggcataa caatgaatgg gctttcctcg gtaccaaatt ccagaaaaga ggcctcccga
     3961 aaggggggcc ttttttcgtt ttggtcccta attgcctatc ttccagtgat ggaacagcat
     4021 ttgtgcattg gctgcaacaa tcagccttac ttgtgcctgt tctatttccg aaccgaccgc
     4081 ttgtatgaat ccatcaaaat tcgttttctc tatgttggat tccttgttgc tcatattgtg
     4141 atgataattt ctacaaatat agtcattggt aactatctat gaaactgttt gatactttta
     4201 ctcatcggat tcttttaata tcgctactct ctacgttcaa ccaaaagaaa taatgactta
     4261 ctgctatatt ttttgcacgt gtggggaaaa tgtggggaaa attcaagcaa aagaaaaagc
     4321 taagtattga actatcaaat acttagcttt ctttcttgta cccagacccc gcatttgaaa
     4381 taattaaagt ggggaaaatg tgggtaaaaa gaaaaatgcg gaaaaacgcc acaattacac
     4441 tgtatttcaa tatgttataa tcctattaaa ttttaatcca agtttaatcg aattgcaaaa
     4501 tatttagcag atgtggggaa aatgctgggg aaaatattta tatttgcagc agagtaaaat
     4561 tcaacctgaa agtctatgaa agtaaccttt atcattaaaa aagcagccaa acgatatgat
     4621 acagaatcca tggctacaat ctatgtccgt tttagaaacg gaaggcagtt agactccgtt
     4681 gctcctactc agttagccat caatcccaat ctatgggatg ataaagacga atgtgtaaaa
     4741 acgaaagctg tctgcaatga agaaatgcgt acccatataa atgaagagat acgccagttg
     4801 aaaacctata tcgagaaggt atatcaacaa gaaaaggaag caatagacaa agaatggcta
     4861 aaaacaacac ttgataaatt ttaccatcct gaaaaatatt ttttgccgga ggaagtggtt
     4921 atcaagccta ccattggaga actattcgat gaatttctaa acaagcaccc tttgtcggaa
     4981 gtacgaaaga aaaatttccg ggttgtcaaa agagccttac tgcgttatga actatatgta
     5041 agggctacaa agagaggaca aaagggcttt atccttgatg tggatttggt aacacctgac
     5101 acgcttcggg atatgtggga tttctttcag aacgaatacc agtattatga actttacccg
     5161 agcatttatg aagccattcc cgaaaagagg acaccacagc ccagaagcaa aaacacgctg
     5221 atagactgtt tttcaagaat acgcacattc ttcctgtggt gcttcgataa caaacgcacc
     5281 acaaacagac ctttcgacaa gtttccgata gaggagtgta catatggtac accttattat
     5341 ataacactcg aagaaaggga caggattttt aatgcagacc tttctgccac cccacaactg
     5401 gcaatacaga gggatatatt catatttcag acactgatag gatgcagggt gagcgacctg
     5461 taccgaatga ccaaactaaa tgtggtcaat gaagccatag aatatattcc caagaaaacc
     5521 aaagagggga atccggttac ggtacgtgtt ccacttaacg acaaagcgaa agaaatcctt
     5581 gaacgctaca aagaatatga gggaaaactg ttgccgttca tatccgagca aaagtacaat
     5641 gatgccataa aaaagatatt caaattagct ggagttgacc gcatcgtaac aatcttagac
     5701 ccgttgacgc acaacgaaat caaacgacct atttatgaag tggcaagcag ccatctggca
     5761 agacgtacgt ttatcggcaa tatctataaa aaagtgaaag acccgaacct tgtttccgca
     5821 ctgtcgggac acaaggaggg aagcaaagct ttcagacgat acagggatat tgacgaagaa
     5881 atgaagaaag accttgtaaa actactggac tgataaaagt atcaaacagt ttcatagata
     5941 gtt
//
