LOCUS       Exported                7531 bp ds-DNA     circular SYN 06-MAR-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7531)
  AUTHORS   Broad Institute
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Mar 11, 2021 from SnapGene 4.3.11
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7531
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    3..27
                     /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)."
     misc_feature    74..109
                     /label=PmcCAST_DR
     misc_feature    111..116
                     /label=BsaI
     misc_feature    120..125
                     /label=BsaI
     misc_feature    127..162
                     /label=PmcCAST_DR
     promoter        270..288
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    289..313
                     /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)."
     CDS             356..1018
                     /codon_start=1
                     /label=PmcCas6
                     /translation="MTAILIQSEEYVDLTFKLRGAPIPLDNGYLTYAALSRICPPLHEL
                     KSIGIHPIAGIPTRNNLLELTAQSRLKIRIYHQQIPLIYPYLAGQAFHIGQNFYQLDIP
                     DYKPLISSESVYSRLVIIKGFQDSTNFIEAVQRQMDNLGIQGKIELLTRQDGTPQRRQL
                     TINKEGKQFKVRGFGVKISELNPEDSLTLQEQGIGGKRKMMCGIFVPATRSKEEEET"
     CDS             1018..2598
                     /codon_start=1
                     /label=PmcCas8
                     /translation="MIVSTQPKISLSLHAADTTIMHRVGMTGLYMTLKRLEKQYPLSRQ
                     RGGHISWFLTADTIELFWEGSDFIALSWLINESFQLDDTGLIHLVGLDNDRIDLRQKIH
                     IHEGICGVFLRLNKFYQAGEIINTELRFEEKQVEYQYKSLTWYAHQTFAEKLCEADTQQ
                     LRHDYIQITSWLYLGGIVRHARTQNTTKLEEKPEYALALLFVPVVCHYCLLHIPSEDLK
                     ERKPHRYLVVIPEIKDFEDASQRRWRLQQLETKQFHVSSLGEAGLLYYSLDDIQPEVAY
                     YQACQVWLYEKTNKASRQRTLMSIEEIKIDKNILITYQQVQKYFKTNYQIIKYKQIFIK
                     VNPIRSLIADNLVKGIHWWSNFWEKLVIEDSKEYLFNQLFSNREGFIIMAENSEEDKQY
                     LIFIKVFQQAMKGNFAKIYAKTEEGKDPPIKKKVERLRAELNYCYDELSFKEYLSDFLV
                     RGGLNKYFNEHQEEIALLIKKSPWQEIRIWSLLAIASYKPKDKLTNRDDSSLSNNQKLE
                     EVNDDSEEE"
     CDS             2576..3547
                     /codon_start=1
                     /label=PmcCas7
                     /translation="MMTQKKNDSNIPNYYLYGTVLTRYGLASLNHDIRRGNKTILQKGY
                     WNNGKIHSFVGSSAIRWALRFYLQKQGYLVNRVWDEEEHINRLTSEDFDPEKFYDDDIF
                     GFALLESAETEEDTSTTKRKKKQTKTSTPNQRMGALGMNMAVSLTPYDGAVKLGAKSGR
                     EKDSTSLHFTEYHATRYQYYFGIDATHLKDFSRILPMIDGIMNLPKVGGSSNIFNYPFC
                     PDSLVFQWTNHFASYISYCFEYCDPKSKEAKLSQEFIDEVECGQIDPSKLWIGGTIVKD
                     LQQLDNFESSPLNKAHIYRNRNEMIEALKTVIKRDLGLEESK"
     CDS             3544..4182
                     /codon_start=1
                     /label=PmcCas5
                     /translation="MIAPLILYLDVPFTTFRESHAREMGKTYPVPPPATVYGMLLSLVG
                     ETNVYRHCGVELAIAMLSSPKKSRILRQMRRFKNADFSHPENVIPCYQEILSNLKCLIW
                     VRSDEEKIQPSLRERIQLAFDHPELVRRFGCLFLGESDQLIKTIKLAREDYLEGVRQWA
                     IRDNRGRLTLPYWVDHVGSRNTRFLRYRIEEMDRLSPPDLAWTMVQSPI"
     terminator      4212..4259
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4430..5221)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(5222..5313)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5361..6099)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     CDS             complement(6309..7391)
                     /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"
     promoter        complement(7392..7469)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(7515..7531,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atggtgagtc gtggctttat gccgttaggc gttgctcaaa gagacctctg
      121 gtctcagtga gtcgtggctt tatgccgtta ggcgttgctc aagatccgaa ttcgagctcg
      181 gcgcgcctgc aggtcgacaa gcttgcggcc gcataatgct taagtcgaac agaaagtaat
      241 cgtattgtac acggccgcat aatcgaaatt aatacgactc actatagggg aattgtgagc
      301 ggataacaat tccccatctt agtatattag ttaagtataa gaaggagata tacatatgac
      361 agctatatta attcaaagtg aggaatatgt agacttaaca tttaagctaa gaggcgcacc
      421 gattcctctt gacaacggtt atcttaccta cgctgcctta tcccgtattt gtcccccact
      481 acatgaacta aagtctattg ggattcatcc gattgctgga ataccgacca ggaacaattt
      541 gcttgagcta actgctcaat ctcgtctaaa aattcggatt tatcatcagc aaattccttt
      601 aatttatcct tatttagcgg gtcaagcttt tcatataggt caaaattttt atcaactaga
      661 tatccccgac tacaaaccgt taatttcttc agaaagtgtc tattccagat tagtgataat
      721 taaagggttc caagactcta ctaactttat tgaagctgtg caacggcaaa tggataattt
      781 aggaatacaa ggaaaaattg aacttcttac ccgacaagat ggaacacctc aacgaagaca
      841 attaaccatc aataaagaag ggaaacagtt taaagttaga ggatttggcg taaaaatcag
      901 tgaactcaat cctgaagatt ccttaacctt gcaagaacag ggaattggcg ggaaacgaaa
      961 gatgatgtgt ggaatatttg tcccagcgac tcgcagcaag gaagaagagg aaacctgatg
     1021 atagtctcta cccaacccaa aatttcccta tctctccatg ctgcggatac gacaataatg
     1081 caccgcgtcg gaatgaccgg actttacatg actttaaagc gattggaaaa gcaatatccc
     1141 ttatctcgtc aacggggagg acatatatca tggtttttga ctgctgatac tattgaatta
     1201 ttctgggaag gaagtgattt catcgcctta tcttggttaa ttaatgagtc ttttcaatta
     1261 gatgatacag gtttaattca tttagtagga ttagacaatg atcgaataga tttaaggcag
     1321 aaaatccata ttcatgaggg aatatgcggt gtttttctac gtcttaataa gttttatcaa
     1381 gcaggagaga taattaacac tgaactaagg tttgaagaaa aacaagtcga gtatcaatat
     1441 aaatccctaa cttggtacgc acatcaaacc tttgcagaga agttatgcga ggcagatacc
     1501 caacaactca gacatgatta cattcaaata accagttggt tgtatttagg gggaattgtt
     1561 cgccatgctc ggacgcagaa tactacaaaa ctagaagaga aacctgaata cgctttagca
     1621 ttattatttg taccagtagt ttgtcattat tgcttacttc atataccatc agaagactta
     1681 aaagaaagga aaccccatcg ctacttggtg gtgattccag aaataaagga ttttgaagac
     1741 gcttctcaaa ggagatggcg attacagcaa ttagaaacta aacagtttca tgttagtagc
     1801 cttggggaag caggattact ttattacagc ttagacgata ttcagcctga agtggcctac
     1861 tatcaagctt gtcaagtttg gttatatgaa aaaacaaata aagcttctcg tcaacggaca
     1921 ttgatgagta tagaagaaat taaaattgat aagaatattt taatcactta tcaacaggtg
     1981 caaaaatact ttaaaactaa ttatcagata attaaatata agcaaatttt tattaaggtc
     2041 aatccgattc gctccctgat tgctgataat ttagtgaaag gaatccattg gtggtctaat
     2101 ttttgggaaa aattggtgat agaggattca aaagaatatt tatttaacca gttattttcc
     2161 aatcgagaag gattcatcat aatggcagag aatagtgaag aagacaagca gtatcttatt
     2221 tttattaagg tatttcagca agctatgaag ggcaactttg ctaaaattta tgccaaaaca
     2281 gaggaaggta aagatcctcc aattaagaag aaagttgaac gattaagggc agagctaaac
     2341 tattgttatg atgaattgtc gtttaaggaa tatttgtctg actttttagt aagaggggga
     2401 ttaaataagt attttaatga gcatcaggaa gagattgcac tactaattaa aaaatcacct
     2461 tggcaagaaa taagaatttg gtcattgtta gcgatcgcca gttataaacc caaagataaa
     2521 ctcactaata gggatgatag ttcattgtca aacaatcaaa aactagaaga agtgaatgat
     2581 gactcagaag aagaatgaca gtaatatccc aaattattat ctttacggaa cagtcctcac
     2641 tcgttatggt ttagcttcct taaatcatga cattagacga ggaaataaga ctattctgca
     2701 aaaaggctat tggaataatg gtaaaattca ttcttttgta ggttcaagtg caattcgttg
     2761 ggcgttacgt ttttatcttc aaaagcaagg ttatttagtt aatcgagtct gggatgagga
     2821 agaacatatt aatcggttaa caagtgaaga tttcgatcca gaaaaatttt atgatgatga
     2881 tatttttggg tttgctttat tagagtctgc tgaaacagag gaggacactt caacaacaaa
     2941 gagaaaaaag aaacaaacaa agaccagcac tcctaatcag cgaatgggtg ctttagggat
     3001 gaatatggct gtttcgctaa cgccttatga tggtgcagtt aagttagggg caaaaagtgg
     3061 cagagagaag gatagtacat ctctccattt tacagaatat catgcaactc gatatcaata
     3121 ttattttgga attgatgcca ctcatctcaa agatttttcg cggattttac ctatgataga
     3181 tgggattatg aatctgccca aggtaggagg gagtagtaat atttttaatt atcctttctg
     3241 tcccgatagt ttggtatttc aatggacaaa tcattttgct tcatatattt cctattgctt
     3301 tgaatattgc gatcctaaga gtaaggaagc aaaactttca caagagttta tagatgaagt
     3361 ggaatgcgga cagattgacc ctagtaaatt gtggattggg ggaacaattg ttaaggattt
     3421 acagcaattg gataattttg aaagttcccc tttgaacaag gcgcatattt atcgtaatcg
     3481 aaatgaaatg attgaagctt tgaaaacagt cattaaaaga gatttgggct tggaagaatc
     3541 aaaatgattg caccattaat tctctatttg gatgttccat ttacgacttt tcgggagtcc
     3601 catgcaaggg aaatgggaaa aacctatcct gttcctcctc cagcgacagt gtatgggatg
     3661 ttgttgtctt tggtaggaga aacgaatgtc tatcgccact gtggggtaga attggccatt
     3721 gcaatgttat ctagccccaa aaagtcgcga attttgcgtc aaatgagacg gtttaagaat
     3781 gctgatttta gccatccaga gaatgttatt ccctgttatc aagaaatttt gtctaatctg
     3841 aagtgtttga tttgggttcg ttctgacgag gaaaagatac aaccaagttt aagggagagg
     3901 atacagttag cgtttgacca tcctgagcta gtaagacggt ttggttgttt atttttggga
     3961 gaaagtgacc agttgattaa aacgatcaaa cttgcaaggg aagattatct agagggagtg
     4021 agacagtggg cgattagaga taatcgaggc aggttaactt taccctattg ggttgaccat
     4081 gtggggtcaa gaaatacccg atttttgagg tatcggattg aggaaatgga tagattatca
     4141 cctcctgatt tagcctggac aatggttcaa tctccgattt gacctaggct gctgccaccg
     4201 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     4261 tgaaacctca ggcatttgag aagcacacgg tcacactgct tccggtagtc aataaaccgg
     4321 taaaccagca atagacataa gcggctattt aacgaccctg ccctgaaccg acgaccgggt
     4381 catcgtggcc ggatcttgcg gcccctcggc ttgaacgaat tgttagacat tatttgccga
     4441 ctaccttggt gatctcgcct ttcacgtagt ggacaaattc ttccaactga tctgcgcgcg
     4501 aggccaagcg atcttcttct tgtccaagat aagcctgtct agcttcaagt atgacgggct
     4561 gatactgggc cggcaggcgc tccattgccc agtcggcagc gacatccttc ggcgcgattt
     4621 tgccggttac tgcgctgtac caaatgcggg acaacgtaag cactacattt cgctcatcgc
     4681 cagcccagtc gggcggcgag ttccatagcg ttaaggtttc atttagcgcc tcaaatagat
     4741 cctgttcagg aaccggatca aagagttcct ccgccgctgg acctaccaag gcaacgctat
     4801 gttctcttgc ttttgtcagc aagatagcca gatcaatgtc gatcgtggct ggctcgaaga
     4861 tacctgcaag aatgtcattg cgctgccatt ctccaaattg cagttcgcgc ttagctggat
     4921 aacgccacgg aatgatgtcg tcgtgcacaa caatggtgac ttctacagcg cggagaatct
     4981 cgctctctcc aggggaagcc gaagtttcca aaaggtcgtt gatcaaagct cgccgcgttg
     5041 tttcatcaag ccttacggtc accgtaacca gcaaatcaat atcactgtgt ggcttcaggc
     5101 cgccatccac tgcggagccg tacaaatgta cggccagcaa cgtcggttcg agatggcgct
     5161 cgatgacgcc aactacctct gatagttgag tcgatacttc ggcgatcacc gcttccctca
     5221 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5281 acatatttga atgtatttag aaaaataaac aaatagctag ctcactcggt cgctacgctc
     5341 cgggcgtgag actgcggcgg gcgctgcgga cacatacaaa gttacccaca gattccgtgg
     5401 ataagcaggg gactaacatg tgaggcaaaa cagcagggcc gcgccggtgg cgtttttcca
     5461 taggctccgc cctcctgcca gagttcacat aaacagacgc ttttccggtg catctgtggg
     5521 agccgtgagg ctcaaccatg aatctgacag tacgggcgaa acccgacagg acttaaagat
     5581 ccccaccgtt tccggcgggt cgctccctct tgcgctctcc tgttccgacc ctgccgttta
     5641 ccggatacct gttccgcctt tctcccttac gggaagtgtg gcgctttctc atagctcaca
     5701 cactggtatc tcggctcggt gtaggtcgtt cgctccaagc tgggctgtaa gcaagaactc
     5761 cccgttcagc ccgactgctg cgccttatcc ggtaactgtt cacttgagtc caacccggaa
     5821 aagcacggta aaacgccact ggcagcagcc attggtaact gggagttcgc agaggatttg
     5881 tttagctaaa cacgcggttg ctcttgaagt gtgcgccaaa gtccggctac actggaagga
     5941 cagatttggt tgctgtgctc tgcgaaagcc agttaccacg gttaagcagt tccccaactg
     6001 acttaacctt cgatcaaacc acctccccag gtggtttttt cgtttacagg gcaaaagatt
     6061 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctactga accgctctag
     6121 atttcagtgc aatttatctc ttcaaatgta gcacctgaag tcagccccat acgatataag
     6181 ttgtaattct catgttagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     6241 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     6301 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     6361 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     6421 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     6481 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     6541 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca
     6601 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     6661 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     6721 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     6781 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     6841 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     6901 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     6961 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     7021 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     7081 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     7141 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     7201 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     7261 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     7321 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     7381 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     7441 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     7501 ctgcattagg aaattaatac gactcactat a
//
