LOCUS       Exported                7402 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses AlRT-Cas1-Cas2 under pT7lac promoter, encodes AlCRISPR 
            array 2 RC, compatible with SENECA acquisition readout.
ACCESSION   .
VERSION     .
KEYWORDS    pFS_0372_pET30-RT-CRISPR_Azospirillum_lipoferum_4B_ARRAY_2_RC
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7402)
  AUTHORS   Schmidt F, Cherepkova MY, Platt RJ
  TITLE     Transcriptional recording by CRISPR spacer acquisition from RNA.
  JOURNAL   Nature. 2018 Oct;562(7727):380-385. doi: 10.1038/s41586-018-0569-1. 
            Epub 2018 Oct 3.
  PUBMED    30283135
REFERENCE   2  (bases 1 to 7402)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7402
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             complement(410..1225)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1133..1152
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1217..1236
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1347..1935
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1836..1855
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2089..2106
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2121..2260
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2186..2205)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2346..2368
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2362..2553)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3362..4444)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4406..4425
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4445..4522)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4728..4747
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4835..4854
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4835..4853
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4854..4878
                     /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)."
     RBS             4893..4915
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(7315..7333)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7329..7376
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ccaggtcttc cgcttcgcaa tctggaacgc ctccggcctc ctcatgacct gttccccagg
       61 ggctagtcgg aagggccatc acgtccaaag gcgcgattga cctgagtttg ttgcccggcc
      121 ggatcgcgcc gcctgcgcgg gacggtcgat ccaagagttt caatccgctc ccaaacgcgg
      181 acaccggcgg aacgtcccgc ggccgctatt cttttgattt ataagggatt ttgccgattt
      241 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
      301 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      361 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
      421 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
      481 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
      541 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
      601 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
      661 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
      721 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
      781 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
      841 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
      901 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
      961 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     1021 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     1081 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     1141 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     1201 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     1261 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     1321 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     1381 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     1441 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     1501 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     1561 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     1621 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     1681 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     1741 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     1801 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     1861 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     1921 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggt cttttgctgg
     1981 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     2041 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     2101 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     2161 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     2221 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     2281 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     2341 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     2401 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     2461 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     2521 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     2581 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     2641 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     2701 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     2761 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     2821 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagatca ttcatgttgt
     2881 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     2941 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     3001 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa
     3061 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     3121 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     3181 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagatag tcataagtgc
     3241 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     3301 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     3361 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     3421 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     3481 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     3541 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     3601 aacatgagct atcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca
     3661 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     3721 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     3781 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     3841 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     3901 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtacca tcttcatggg
     3961 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     4021 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     4081 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     4141 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     4201 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     4261 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     4321 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     4381 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     4441 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     4501 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     4561 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     4621 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     4681 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     4741 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     4801 tagaggatcg agatcgatct cgatcccgcg aaattaatac gactcactat aggggaattg
     4861 tgagcggata acaattcccc tctagaaata attttgttta actttaagaa ggagatatac
     4921 atatgcctac ggcaccccct gataatgcag acctatttga agaagtgacc agattggaca
     4981 cacttgagag agcttggggt agagtcttgc gaaatgctgg tgctgctggt ggtgacggtt
     5041 tgactgtggg tcgttttgca gaagctgctc catctaggct tttggcatta catcgtactc
     5101 ttagaatggg tgactatagg cctggtcccc ttagaagatt atctattcca aaaccagatg
     5161 gtgctttaag accactggct atcccaccag tcactgatcg tgtcgctcaa actgctgctg
     5221 ctcttgtatt gacgccactt ttggacggag aatttgagga cgcttcattt gggtatcgac
     5281 ctggcagatc agttcctcaa gcagtagcca gggtagctag atggagagat caaggatacg
     5341 attgggtcgt ggatgcagat attgagagat actttgagcg tgttccacat gatagacttc
     5401 taatacgatt ggaaaggagt attggtgctg gtccacttac tgaattgatt gcagtgtggt
     5461 tagagtcggg agccgaaaat ggtgttggtt tacctcaagg ttctccatta agtccattac
     5521 tatctaattt gtatcttgat gacttagatg aagctcttga tggcagaggt ctacgtcttg
     5581 ttaggtttgc agatgacttt gttctattgt gtagaagcag agaacgtgct gagagagctt
     5641 tagatcacgc agctgctgta ttggaagaac acggtttgag actgaataga gataaaacca
     5701 gaattgttcc ctttgatcaa ggatttaggt tcttgggtca cctatttgta agatccttgg
     5761 ttttgccttc accaagacca gacgatggtg aagaaaccga aggtgacgca ttattgagag
     5821 cactcgcaat tagagatgcc gccgctgaag cagaagaagc tgccgcagaa gaaagagata
     5881 ggcaaagtcg tgctgctggg cttgatcctg cacttagaac gttacacgta caaacgccta
     5941 gaaggagact agctttaagg aatgaggctt ttactgttgt tgagagggat gaccttggtg
     6001 gtgaaagaga actcattgct attcatcatc atcatttgga tcgtattgat ctaggcccag
     6061 aagcagatgc agacgctgaa gcattgagat gggcccttgc tacagatact gagttggcat
     6121 ttgttgatgg ccacggtgct actcacggta gactaacgag gcctgaagct aggagagctg
     6181 ctttgcattt agaccaagcc cgtcatgctc ttgatgaagg tttgcgttta gatctagcaa
     6241 gaagaattgt agatggaaga ttgcgaaatc aaagagcact tcttagaaga ttgaatcgtt
     6301 ccagaaaact tggagttgtc gaggacgctg tcctcgctat aaatgcacta cttagaagat
     6361 tagatacagc tgctgatgtt gctgcattgt taggatttga aggtcaggga gctgctcgat
     6421 actggccagc tttagctgcc caaattgaag gagagtggga atttgaagga agaaggcgta
     6481 gaccaccccc tgaccctgta aatgctgtac tctcttacct ctcgggttta ttggagagag
     6541 atgtagctgc tcttattgct aggcatgggc tacatccggg atttggagta ctacattcac
     6601 cacaagatcg acatgatgca ggaatttatg atttgatgga agaatttaga gcccccttaa
     6661 tggaaggcct ggctgttact ttgtttaata ggagaacgct caggccagat cactttagta
     6721 gacgtgaaac gggtgaggga gaaattaaag gatgtcgtat cgaccccgat gctgttggag
     6781 ctattattag ggcttacgag caatgggtcc gtcgtcctgt tgcatctcca agagatggta
     6841 aaagaactac ttggagaggt cttataggat tccaagcaca agcattggct gcacacgtcc
     6901 aaggaaggga accctataga gcttacgtga tggactatta gcgtacgtta actttaagaa
     6961 ggagaactta agatgtctga aacaccaatg gttatggtgt tctgctacga tgtcgccaat
     7021 gatagaagaa gaaggagagt ctcagcagta cttgaggagt ggggagttag ggtacaaaag
     7081 tccgtatttg aagcaagact aactgagcct caagctagag ctttgttagc aagagctgga
     7141 aaagaactgg gtccaagaga ttctcttaga atgtatgcac ttagcgcaca tggcctcgct
     7201 cattctgctg ccctcggtgg ggctccagtt cccgagcaac aagacttttg gctcttgtaa
     7261 ctcgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     7321 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     7381 aaggaggaac tatatccgga ta
//
