LOCUS       Exported                7267 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses group II intron Ll.LtrB and IEP (LtrA) from Lactoccus 
            lactis under control of T7 promoter. Ll.LtrB is retargeted to insert
            into 635s locus of LacZ gene and ura3 gene is cloned inside..
ACCESSION   .
VERSION     .
KEYWORDS    pACDura3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7267)
  AUTHORS   Velazquez E, Lorenzo V, Al-Ramahi Y
  TITLE     Recombination-Independent Genome Editing through 
            CRISPR/Cas9-Enhanced TargeTron Delivery.
  JOURNAL   ACS Synth Biol. 2019 Sep 20;8(9):2186-2193. doi: 
            10.1021/acssynbio.9b00293. Epub 2019 Sep 3.
  PUBMED    31419111
REFERENCE   2  (bases 1 to 7267)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7267
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     134..153
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(220..322)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(714..731)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(848..1393)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(928..947)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1638..1657
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1745..1764
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1745..1763
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1764..1788
                     /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)."
     promoter        2528..2575
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     RBS             3741..3763
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     terminator      5939..6025
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6117..6144
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(6827..219)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
ORIGIN
        1 gaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt
       61 gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt
      121 ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga
      181 tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga
      241 aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt
      301 ggaacctctt acgtgccgat caacgtctca ttttcgccaa aagttggccc agggcttccc
      361 ggtatcaaca gggacaccag gatttattta ttctgcgaag tgatcttccg tcacaggtat
      421 ttattcggcg caaagtgcgt cgggtgatgc tgccaactta ctgatttagt gtatgatggt
      481 gtttttgagg tgctccagtg gcttctgttt ctatcagctg tccctcctgt tcagctactg
      541 acggggtggt gcgtaacggc aaaagcaccg ccggacatca gcgctagcgg agtgtatact
      601 ggcttactat gttggcactg atgagggtgt cagtgaagtg cttcatgtgg caggagaaaa
      661 aaggctgcac cggtgcgtca gcagaatatg tgatacagga tatattccgc ttcctcgctc
      721 actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta cgaacggggc
      781 ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa
      841 agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc
      901 agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggcggctccc
      961 tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc
     1021 gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac
     1081 tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt
     1141 gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg taattgattt
     1201 agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg
     1261 tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt
     1321 cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc
     1381 aaaacgatct caagaagatc atcttattaa tcagataaaa tatttctagc tagatttcag
     1441 tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat aagttgtaat
     1501 tctcatgttt gacagcttat catcgataag ctcaaggaga tggcgcccaa cagtcccccg
     1561 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     1621 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     1681 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatcgatct cgatcccgcg
     1741 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaaaa
     1801 agcttataat tatccttaca tttcccgtga gtgcgcccag atagggtgtt aagtcaagta
     1861 gtttaaggta ctactctgta agataacaca gaaaacagcc aacctaaccg aaaagcgaaa
     1921 gctgatacgg gaacagagca cggttggaaa gcgatgagtt acctaaagac aatcgggtac
     1981 gactgagtcg caatgttaat cagatataag gtataagttg tgtttactga acgcaagttt
     2041 ctaatttcgg ttaaatgtcg atagaggaaa gtgtctgaaa cctctagtac aaagaaaggt
     2101 aagttacgtc acgggactta tctgttatca ccacatttgt acaatctgta ggagaaccta
     2161 tgggaacgaa acgaaagcga tgccgagaat ctgaatttac caagacttaa cactaactgg
     2221 ggatacccta aacaagaatg cctaatagaa aggaggaaaa aggctatagc actagagctt
     2281 gaaaatcttg caagggtacg gagtactcgt agtagtctga gaagggtaac gccctttaca
     2341 tggcaaaggg gtacagttat tgtgtactaa aattaaaaat tgattaggga ggaaaacctc
     2401 aaaatgaaac caacaatggc aattttagaa agaatcagta aaaattcaca agaaaatata
     2461 gacgaagttt ttacaagact ttatcgttat cttttacgtc cagatattta ttacgtggcg
     2521 acgcgttgtt gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt
     2581 gaggaactaa acccctacgt agaggatcga gatctcgatc ccgcgaaatg agaccacaac
     2641 ggtttcccta aagttaagga ggaaaaacat atgtccaagg ctacttacaa ggagcgtgct
     2701 gctacccacc cttctccggt tgctgcaaaa ctgttcaaca ttatgcacga gaagcagacg
     2761 aacctgtgcg cttccctgga tgtccgtact actaaggagc tgctggagct ggttgaagca
     2821 ctgggtccga aaatttgcct gctgaaaact cacgtagaca tcctgactga cttcagcatg
     2881 gaaggtaccg tcaaaccgct gaaagccctg agcgcaaaat acaacttcct gctgttcgaa
     2941 gaccgtaagt tcgctgacat cggtaacacc gtgaaactgc agtacagcgc gggtgtgtac
     3001 cgtatcgccg aatgggcaga catcacgaac gcacatggtg tggtgggtcc aggtatcgtt
     3061 tctggtctga aacaggcagc tgaagaagtg accaaagaac cgcgtggtct gctgatgctg
     3121 gcggaactgt cttgtaaagg ttccctggcg accggtgaat ataccaaagg taccgttgac
     3181 atcgcgaaat ccgacaaaga cttcgttatt ggctttatcg cgcagcgtga catgggcggc
     3241 cgtgacgaag gttacgattg gctgattatg acgccgggtg ttggtctgga tgataaaggc
     3301 gatgccctgg gccaacaata ccgtaccgta gatgatgtag tttctaccgg ctctgatatt
     3361 atcatcgttg gccgcggcct gtttgccaaa ggccgcgatg cgaaagtaga aggcgaacgt
     3421 tatcgcaaag cgggctggga agcgtatctg cgccgctgtg gccagcagaa ttaataaact
     3481 tgcacgcgtt gggaaatggc aatgatagcg aaacaacgta aaactcttgt tgtatgcttt
     3541 cattgtcatc gtcacgtgat tcataaacac aagtgaatgt cgacagtgaa tttttacgaa
     3601 cgaacaataa cagagccgta tactccgaga ggggtacgta cggttcccga agagggtggt
     3661 gcaaaccagt cacagtaatg tgaacaaggc ggtacctccc tacttcacca tatcattttc
     3721 tgcagccccc tagaaataat tttgtttaac tttaagaagg agatatacat atatggctag
     3781 atcgtccatt ccgacagcat cgccagtcac tatggcgtgc tgctagcgct atatgcgttg
     3841 atgcaatttc tatgcactcg tagtagtctg agaagggtaa cgccctttac atggcaaagg
     3901 ggtacagtta ttgtgtacta aaattaaaaa ttgattaggg aggaaaacct caaaatgaaa
     3961 ccaacaatgg caattttaga aagaatcagt aaaaattcac aagaaaatat agacgaagtt
     4021 tttacaagac tttatcgtta tcttttacgt ccagatattt attacgtggc gtatcaaaat
     4081 ttatattcca ataaaggagc ttccacaaaa ggaatattag atgatacagc ggatggcttt
     4141 agtgaagaaa aaataaaaaa gattattcaa tctttaaaag acggaactta ctatcctcaa
     4201 cctgtacgaa gaatgtatat tgcaaaaaag aattctaaaa agatgagacc tttaggaatt
     4261 ccaactttca cagataaatt gatccaagaa gctgtgagaa taattcttga atctatctat
     4321 gaaccggtat tcgaagatgt gtctcacggt tttagacctc aacgaagctg tcacacagct
     4381 ttgaaaacaa tcaaaagaga gtttggcggc gcaagatggt ttgtggaggg agatataaaa
     4441 ggctgcttcg ataatataga ccacgttaca ctcattggac tcatcaatct taaaatcaaa
     4501 gatatgaaaa tgagccaatt gatttataaa tttctaaaag caggttatct ggaaaactgg
     4561 cagtatcaca aaacttacag cggaacacct caaggtggaa ttctatctcc tcttttggcc
     4621 aacatctatc ttcatgaatt ggataagttt gttttacaac tcaaaatgaa gtttgaccga
     4681 gaaagtccag aaagaataac acctgaatat cgggagctcc acaatgagat aaaaagaatt
     4741 tctcaccgtc tcaagaagtt ggagggtgaa gaaaaagcta aagttctttt agaatatcaa
     4801 gaaaaacgta aaagattacc cacactcccc tgtacctcac agacaaataa agtattgaaa
     4861 tacgtccggt atgcggacga cttcattatc tctgttaaag gaagcaaaga ggactgtcaa
     4921 tggataaaag aacaattaaa actttttatt cataacaagc taaaaatgga attgagtgaa
     4981 gaaaaaacac tcatcacaca tagcagtcaa cccgctcgtt ttctgggata tgatatacga
     5041 gtaaggagat ctggaacgat aaaacgatct ggtaaagtca aaaagagaac actcaatggg
     5101 agtgtagaac tccttattcc tcttcaagac aaaattcgtc aatttatttt tgacaagaaa
     5161 atagctatcc aaaagaaaga tagctcatgg tttccagttc acaggaaata tcttattcgt
     5221 tcaacagact tagaaatcat cacaatttat aattctgaac tccgcgggat ttgtaattac
     5281 tacggtctag caagtaattt taaccagctc aattattttg cttatcttat ggaatacagc
     5341 tgtctaaaaa cgatagcctc caaacataag ggaacacttt caaaaaccat ttccatgttt
     5401 aaagatggaa gtggttcgtg ggggatcccg tatgagataa agcaaggtaa gcagcgccgt
     5461 tattttgcaa attttagtga atgtaaatcc ccttatcaat ttacggatga gataagtcaa
     5521 gctcctgtat tgtatggcta tgcccggaat actcttgaaa acaggttaaa agctaaatgt
     5581 tgtgaattat gtgggacgtc tgatgaaaat acttcctatg aaattcacca tgtcaataag
     5641 gtcaaaaatc ttaaaggcaa agaaaaatgg gaaatggcaa tgatagcgaa acaacgtaaa
     5701 actcttgttg tatgctttca ttgtcatcgt cacgtgattc ataaacacaa gtgaatgtcg
     5761 agcacccgtt ctcggagcac tgtccgaccg ctttggccgc cgcccagtcc tgctcgcttc
     5821 gctacttgga gccactatcg actacgcgat catggcgacc acacccgtcc tgtggatcgc
     5881 caagccgccg atggtagtgt ggggtctccc catgcgagag tagggaactg ccaggcatca
     5941 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt
     6001 gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg
     6061 gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa
     6121 ggccatcctg acggatggcc tttttgcgtt tctacaaact cttcctgtcg tcatatctac
     6181 aagccatccc cccacagata cggtaaacta gcctcgtttt tgcatcagga aagcagaacg
     6241 ccatgagcgg cctcatttct tattctgagt tacaacagtc cgcaccgctg ccggtagctc
     6301 cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat
     6361 gcaactcgta ggacaggtgc cggcagcgcc caacagtccc ccggccacgg ggcctgccac
     6421 catacccacg ccgaaacaag cgccctgcac cattatgttc cggatctgca tcgcaggatg
     6481 ctgctggcta ccctgtggaa cacctacatc tgtattaacg aagcgctaac cgtttttatc
     6541 aggctctggg aggcagaata aatgatcata tcgtcaatta ttacctccac ggggagagcc
     6601 tgagcaaact ggcctcaggc atttgagaag cacacggtca cactgcttcc ggtagtcaat
     6661 aaaccggtaa accagcaata gacataagcg gctatttaac gaccctgccc tgaaccgacg
     6721 accgggtcga atttgctttc gaatttctgc cattcatccg cttattatca cttattcagg
     6781 cgtagcaacc aggcgtttaa gggcaccaat aactgcctta aaaaaattac gccccgccct
     6841 gccactcatc gcagtactgt tgtaattcat taagcattct gccgacatgg aagccatcac
     6901 aaacggcatg atgaacctga atcgccagcg gcatcagcac cttgtcgcct tgcgtataat
     6961 atttgcccat ggtgaaaacg ggggcgaaga agttgtccat attggccacg tttaaatcaa
     7021 aactggtgaa actcacccag ggattggctg agacgaaaaa catattctca ataaaccctt
     7081 tagggaaata ggccaggttt tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa
     7141 actgccggaa atcgtcgtgg tattcactcc agagcgatga aaacgtttca gtttgctcat
     7201 ggaaaacggt gtaacaaggg tgaacactat cccatatcac cagctcaccg tctttcattg
     7261 ccatacg
//
