LOCUS       Exported                5672 bp ds-DNA     circular SYN 17-NOV-2018
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5672)
  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..5672
                     /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=oMT25
     primer_bind     2968..2987
                     /label=pNBU1_seq.FOR
     primer_bind     complement(3045..3062)
                     /label=oMT1013
     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..3136)
                     /label=oMT1185
     primer_bind     3123..3150
                     /label=oMT1186
     misc_feature    3123..3130
                     /label=L3S3P21
     misc_feature    3130..3133
                     /label=CGCT
     misc_feature    3131..3649
                     /label=Amp up this region
     CDS             complement(join(3204..3208,3210..3249,3251..3254,
                     3261..3272))
                     /codon_start=1
                     /label=LacZ alpha
                     /translation="WRNSAPPIALPNSCAA*MAM"
     CDS             complement(join(3204..3208,3210..3249,3251..3272))
                     /label=LacZ alpha
     misc_feature    3231..3249
                     /label=spacer:lacZ
     misc_feature    complement(3255..3260)
                     /label=<-- EarI site
     primer_bind     3343..3360
                     /label=M13-fwd
     primer_bind     complement(3426..3446)
                     /label=M13-rev
     misc_binding    complement(3452..3474)
                     /label=LacO
     misc_feature    complement(3477..3506)
                     /label=3' lacI KO check - R (580bp)
     misc_feature    complement(3479..3484)
                     /label=pLac -10 box
     misc_feature    complement(3503..3508)
                     /label=pLac -35 box
     misc_feature    complement(3527..3541)
                     /label=CAP site
     misc_feature    complement(3544..3564)
                     /label=LacO3
     misc_feature    complement(3554)
                     /label=Center LacO3
     misc_feature    complement(3557..3564)
                     /label=LacI
     primer_bind     complement(3615..3650)
                     /label=oMT1187
     primer_bind     3633..3664
                     /label=oMT1188
     misc_feature    3640..3643
                     /label=CAAT
     misc_feature    3650..3655
                     /label=problematic assembly region
     terminator      3656..3716
                     /label=L3S2P21 Terminator
     primer_bind     complement(3695..3735)
                     /label=oMT1272
     misc_feature    3695..3716
                     /label=Feature 67
     primer_bind     3717..3744
                     /label=oMT1259
     primer_bind     complement(3842..3862)
                     /label=pNBU1_seq.REV
     misc_feature    complement(3891..3929)
                     /label=mmD660
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    3930..5672
                     /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    3930..4729
                     /label=Piece1
     misc_feature    3930..3959
                     /label=Geneious type: Editing History Insertion
                     /note="Geneious type: Editing History Insertion"
     primer_bind     3930..3958
                     /label=mmD658
     primer_bind     3951..3971
                     /label=oMT113
     misc_feature    3960..4289
                     /label=attN1 Region
     primer_bind     3960..3979
                     /label=intN1 Construct PCR Product (fwd) - pNBU2-PcfxA
                     /label=intN1 Construct PCR Product (fwd) - 
                     pNBU2-PcfxA-NanoLuc PCR Product
     misc_feature    4010..4025
                     /label=DR1
     misc_feature    4043..4057
                     /label=IR1
     misc_feature    4067..4081
                     /label=IR2
     misc_feature    4087..4100
                     /label=CC Region
     misc_feature    4099
                     /label=G-->C
     misc_feature    4099
                     /label=G
                     /note="Geneious type: Editing History Replacement"
     misc_feature    4100
                     /label='-1'
     misc_feature    4101
                     /label='+1'
     misc_feature    4118..4133
                     /label=DR2
     primer_bind     complement(4156..4178)
                     /label=oMT114
     misc_feature    4242..4258
                     /label=DR3
     CDS             4305..4729
                     /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            4305..4729
                     /gene="intN1"
                     /label=intN1
                     /label=intN1 gene
     primer_bind     complement(4521..4548)
                     /label=oMT157
     primer_bind     4521..4548
                     /label=oMT156
     misc_feature    complement(4699..4729)
                     /label=mmD659
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    4700..5672
                     /label=Piece2
     CDS             4700..5642
                     /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            4700..5642
                     /gene="intN1"
                     /label=intN1
                     /label=intN1 gene
     misc_feature    4700..4729
                     /label=Piece2
     misc_feature    4700..4729
                     /label=Ligation
     primer_bind     4700..4719
                     /label=intN1 Construct PCR Product 2 (fwd)
     misc_feature    complement(4705..4729)
                     /label=intN1 Construct PCR Product (rev)
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(5618..5644)
                     /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    5634
                     /label=T
                     /note="Geneious type: Editing History Replacement"
     misc_feature    complement(5636..5672)
                     /label=mmD661
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    5636
                     /label=A
                     /note="Geneious type: Editing History Replacement"
     misc_feature    5643..5672
                     /label=HOMOLOGY
     misc_feature    5645..5672
                     /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 gcttctgtct tctgcaccat atgcggtgtg aaataccgca cagatgcgta
     3181 aggagaaaat accgcatcag gcgccattag ccattcaggc tgcgcaactg ttgggaaggg
     3241 cgatcggtgt gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg
     3301 cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt
     3361 gaattcgagc tcggtacccg gggatcctct agagtcgacc tgcaggcatg caagcttggc
     3421 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     3481 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     3541 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     3601 ttaatgaatc ggccaacgcg cggggaagac agggtctccc aatgaatggg ctttcctcgg
     3661 taccaaattc cagaaaagag gcctcccgaa aggggggcct tttttcgttt tggtccctaa
     3721 ttgcctatct tccagtgatg gaacagcatt tgtgcattgg ctgcaacaat cagccttact
     3781 tgtgcctgtt ctatttccga accgaccgct tgtatgaatc catcaaaatt cgttttctct
     3841 atgttggatt ccttgttgct catattgtga tgataatttc tacaaatata gtcattggta
     3901 actatctatg aaactgtttg atacttttac tcatcggatt cttttaatat cgctactctc
     3961 tacgttcaac caaaagaaat aatgacttac tgctatattt tttgcacgtg tggggaaaat
     4021 gtggggaaaa ttcaagcaaa agaaaaagct aagtattgaa ctatcaaata cttagctttc
     4081 tttcttgtac ccagaccccg catttgaaat aattaaagtg gggaaaatgt gggtaaaaag
     4141 aaaaatgcgg aaaaacgcca caattacact gtatttcaat atgttataat cctattaaat
     4201 tttaatccaa gtttaatcga attgcaaaat atttagcaga tgtggggaaa atgctgggga
     4261 aaatatttat atttgcagca gagtaaaatt caacctgaaa gtctatgaaa gtaaccttta
     4321 tcattaaaaa agcagccaaa cgatatgata cagaatccat ggctacaatc tatgtccgtt
     4381 ttagaaacgg aaggcagtta gactccgttg ctcctactca gttagccatc aatcccaatc
     4441 tatgggatga taaagacgaa tgtgtaaaaa cgaaagctgt ctgcaatgaa gaaatgcgta
     4501 cccatataaa tgaagagata cgccagttga aaacctatat cgagaaggta tatcaacaag
     4561 aaaaggaagc aatagacaaa gaatggctaa aaacaacact tgataaattt taccatcctg
     4621 aaaaatattt tttgccggag gaagtggtta tcaagcctac cattggagaa ctattcgatg
     4681 aatttctaaa caagcaccct ttgtcggaag tacgaaagaa aaatttccgg gttgtcaaaa
     4741 gagccttact gcgttatgaa ctatatgtaa gggctacaaa gagaggacaa aagggcttta
     4801 tccttgatgt ggatttggta acacctgaca cgcttcggga tatgtgggat ttctttcaga
     4861 acgaatacca gtattatgaa ctttacccga gcatttatga agccattccc gaaaagagga
     4921 caccacagcc cagaagcaaa aacacgctga tagactgttt ttcaagaata cgcacattct
     4981 tcctgtggtg cttcgataac aaacgcacca caaacagacc tttcgacaag tttccgatag
     5041 aggagtgtac atatggtaca ccttattata taacactcga agaaagggac aggattttta
     5101 atgcagacct ttctgccacc ccacaactgg caatacagag ggatatattc atatttcaga
     5161 cactgatagg atgcagggtg agcgacctgt accgaatgac caaactaaat gtggtcaatg
     5221 aagccataga atatattccc aagaaaacca aagaggggaa tccggttacg gtacgtgttc
     5281 cacttaacga caaagcgaaa gaaatccttg aacgctacaa agaatatgag ggaaaactgt
     5341 tgccgttcat atccgagcaa aagtacaatg atgccataaa aaagatattc aaattagctg
     5401 gagttgaccg catcgtaaca atcttagacc cgttgacgca caacgaaatc aaacgaccta
     5461 tttatgaagt ggcaagcagc catctggcaa gacgtacgtt tatcggcaat atctataaaa
     5521 aagtgaaaga cccgaacctt gtttccgcac tgtcgggaca caaggaggga agcaaagctt
     5581 tcagacgata cagggatatt gacgaagaaa tgaagaaaga ccttgtaaaa ctactggact
     5641 gataaaagta tcaaacagtt tcatagatag tt
//
