LOCUS       sequence-356878-        7504 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses eSpCas9 and gRNA that targets ColE1 plasmids for curing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 2903 to 2937)
  AUTHORS   Lee KZ, Mechikoff MA, Parasa MK, Rankin TJ, Pandolfi P, Fitzgerald 
            KS, Hillman ET, Solomon KV
  TITLE     Repurposing the Homing Endonuclease I-SceI for Positive Selection 
            and Development of Gene-Editing Technologies.
  JOURNAL   ACS Synth Biol. 2022 Jan 21;11(1):53-60. doi: 
            10.1021/acssynbio.1c00340. Epub 2022 Jan 10.
  PUBMED    35007422
REFERENCE   2  (bases 1 to 7504)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/acssynbio.1c00340"; journalName: "ACS Synth Biol"; date: 
            "2022-01-21- 21"; volume: "11"; issue: "1"; pages: "53-60"
COMMENT     Sequence Label: pFREE2-eSpCas9-sgRNA
FEATURES             Location/Qualifiers
     source          1..7504
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(35..53)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      49..96
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             230..853
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(303..322)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             complement(1015..1830)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1015..1830)
                     /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     1738..1757
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1831..1922)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1940..2575)
                     /direction=LEFT
                     /label=ColA ori
                     /note="Plasmids containing the ColA origin of replication 
                     can be propagated in E. coli cells that contain additional 
                     plasmids with compatible origins."
     primer_bind     complement(2085..2104)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_RNA        complement(2824..2902)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     promoter        complement(2903..2937)
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     repeat_region   3080..3115
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   3146..3181
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     protein_bind    3255..3273
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3280..3298
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             3392..7498
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
ORIGIN
        1 gatcccatgt taccggtatc ctaggctgct gccaccgctg agcaataact agcataaccc
       61 cttggggcct ctaaacgggt cttgaggggt tttttgctga aacctcaggc atttgagaag
      121 cacacggtca cactgcttcc ggtagtcaat aaaccggtat tgacgccaac gggtcacacg
      181 ggtacagtgc tagcagaaac atatccatga aatcccgccc cgaattcata tgtctagatt
      241 agataaaagt aaagtgatta acagcgcatt agagctgctt aatgaggtcg gaatcgaagg
      301 tttaacaacc cgtaaactcg cccagaagct aggtgtagag cagcctacat tgtattggca
      361 tgtaaaaaat aagcgggctt tgctcgacgc cttagccatt gagatgttag ataggcacca
      421 tactcacttt tgccctttag aaggggaaag ctggcaagat tttttacgta ataacgctaa
      481 aagttttaga tgtgctttac taagtcatcg cgatggagca aaagtacatt taggtacacg
      541 gcctacagaa aaacagtatg aaactctcga aaatcaatta gcctttttat gccaacaagg
      601 tttttcacta gagaatgcat tatatgcact cagcgctgtg gggcatttta ctttaggttg
      661 cgtattggaa gatcaagagc atcaagtcgc taaagaagaa agggaaacac ctactactga
      721 tagtatgccg ccattattac gacaagctat cgaattattt gatcaccaag gtgcagagcc
      781 agccttctta ttcggccttg aattgatcat atgcggatta gaaaaacaac ttaaatgtga
      841 aagtgggtct taagcaatag acataagcgg ctatttaacg accctgccct gaaccgacga
      901 caagctgacg accgggtctc cgcaagtggc acttttcggg gaaatgtgcg cggaacccct
      961 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
     1021 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     1081 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     1141 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     1201 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     1261 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     1321 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     1381 agcgagacga aatacgcggt cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     1441 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     1501 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     1561 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     1621 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     1681 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     1741 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     1801 gcaagacgtt tcccgttgaa tatggctcat actcttcctt tttcaatatt attgaagcat
     1861 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     1921 aataggcatg ctagcgcaga aacgtcctag aagatgccag gaggatactt agcagagaga
     1981 caataaggcc ggagcgaagc cgtttttcca taggctccgc ccccctgacg aacatcacga
     2041 aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     2101 tccccctgat ggctccctct tgcgctctcc tgttcccgtc ctgcggcgtc cgtgttgtgg
     2161 tggaggcttt acccaaatca ccacgtcccg ttccgtgtag acagttcgct ccaagctggg
     2221 ctgtgtgcaa gaaccccccg ttcagcccga ctgctgcgcc ttatccggta actatcatct
     2281 tgagtccaac ccggaaagac acgacaaaac gccactggca gcagccattg gtaactgaga
     2341 attagtggat ttagatatcg agagtcttga agtggtggcc taacagaggc tacactgaaa
     2401 ggacagtatt tggtatctgc gctccactaa agccagttac caggttaagc agttccccaa
     2461 ctgacttaac cttcgatcaa accgcctccc caggcggttt tttcgtttac agagcaggag
     2521 attacgacga tcgtaaaagg atctcaagaa gatcctttac ggattcccga caccatcact
     2581 ctagatttca gtgcaattta tctcttcaaa tgtagcacct gaagtcagcc ccatacgata
     2641 taagttgtaa ttctcatgtt agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     2701 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     2761 attgcgttgc gcacaccgac tagcgaaaaa accccgccga agcggggttt tttgcgaaaa
     2821 aaagcaccga ctcggtgcca ctttttcaag ttgataacgg actagcctta ttttaacttg
     2881 ctatgctgtt ttgaatggtt ccgctagcac tgtacctagg actgagctag ccgtcaaaag
     2941 cagcgtatat accactgcag tggccacaat tcagcaaatt gtgaacatca tcacgttcat
     3001 ctttccctgg ttgccaatgg cccattttcc tgtcagtaac gagaaggtcg cgaattcagg
     3061 cgctttttag actggtcgtg ttttagagct atgctgtttt gaatggtccc aaaactgcgg
     3121 cgagcggtat cagctcactc aaagggtttt agagctatgc tgttttgaat ggtcccaaaa
     3181 caaaaaaaaa ccccgcccct gacagggcgg ggtttttttt ttaattaaag tctggtcctc
     3241 gagtctggtt ataatcccta tcagtgatag agattgacat ccctatcagt gatagagata
     3301 ctgagcacag aaggagatat acatggataa gaaatactga gcactatcag tgataggtcg
     3361 acagatactg agcacagaag gagatataca tatggataag aaatactcaa taggcttaga
     3421 tatcggcaca aatagcgtcg gatgggcggt gatcactgat gaatataagg ttccgtctaa
     3481 aaagttcaag gttctgggaa atacagaccg ccacagtatc aaaaaaaatc ttataggggc
     3541 tcttttattt gacagtggag agacagcgga agcgactcgt ctcaaacgga cagctcgtag
     3601 aaggtataca cgtcggaaga atcgtatttg ttatctacag gagatttttt caaatgagat
     3661 ggcgaaagta gatgatagtt tctttcatcg acttgaagag tcttttttgg tggaagaaga
     3721 caagaagcat gaacgtcatc ctatttttgg aaatatagta gatgaagttg cttatcatga
     3781 gaaatatcca actatctatc atctgcgaaa aaaattggta gattctactg ataaagcgga
     3841 tttgcgctta atctatttgg ccttagcgca tatgattaag tttcgtggtc attttttgat
     3901 tgagggagat ttaaatcctg ataatagtga tgtggacaaa ctatttatcc agttggtaca
     3961 aacctacaat caattatttg aagaaaaccc tattaacgca agtggagtag atgctaaagc
     4021 gattctttct gcacgattga gtaaatcaag acgattagaa aatctcattg ctcagctccc
     4081 cggtgagaag aaaaatggct tatttgggaa tctcattgct ttgtcattgg gtttgacccc
     4141 taattttaaa tcaaattttg atttggcaga agatgctaaa ttacagcttt caaaagatac
     4201 ttacgatgat gatttagata atttattggc gcaaattgga gatcaatatg ctgatttgtt
     4261 tttggcagct aagaatttat cagatgctat tttactttca gatatcctaa gagtaaatac
     4321 tgaaataact aaggctcccc tatcagcttc aatgattaaa cgctacgatg aacatcatca
     4381 agacttgact cttttaaaag ctttagttcg acaacaactt ccagaaaagt ataaagaaat
     4441 cttttttgat caatcaaaaa acggatatgc aggttatatt gatgggggag ctagccaaga
     4501 agaattttat aaatttatca aaccaatttt agaaaaaatg gatggtactg aggaattatt
     4561 ggtgaaacta aatcgtgaag atttgctgcg caagcaacgg acctttgaca acggctctat
     4621 tccccatcaa attcacttgg gtgagctgca tgctattttg agaagacaag aagactttta
     4681 tccattttta aaagacaatc gtgagaagat tgaaaaaatc ttgacttttc gaattcctta
     4741 ttatgttggt ccattggcgc gtggcaatag tcgttttgca tggatgactc ggaagtctga
     4801 agaaacaatt accccatgga attttgaaga agttgtcgat aaaggtgctt cagctcaatc
     4861 atttattgaa cgcatgacaa actttgataa aaatcttcca aatgaaaaag tactaccaaa
     4921 acatagtttg ctttatgagt attttacggt ttataacgaa ttgacaaagg tcaaatatgt
     4981 tactgaagga atgcgaaaac cagcatttct ttcaggtgaa cagaagaaag ccattgttga
     5041 tttactcttc aaaacaaatc gaaaagtaac cgttaagcaa ttaaaagaag attatttcaa
     5101 aaaaatagaa tgttttgata gtgttgaaat ttcaggagtt gaagatagat ttaatgcttc
     5161 attaggtacc taccatgatt tgctaaaaat tattaaagat aaagattttt tggataatga
     5221 agaaaatgaa gatatcttag aggatattgt tttaacattg accttatttg aagataggga
     5281 gatgattgag gaaagactta aaacatatgc tcacctcttt gatgataagg tgatgaaaca
     5341 gcttaaacgt cgccgttata ctggttgggg acgtttgtct cgaaaattga ttaatggtat
     5401 tagggataag caatctggca aaacaatatt agattttttg aaatcagatg gttttgccaa
     5461 tcgcaatttt atgcagctga tccatgatga tagtttgaca tttaaagaag acattcaaaa
     5521 agcacaagtg tctggacaag gcgatagttt acatgaacat attgcaaatt tagctggtag
     5581 ccctgctatt aaaaaaggta ttttacagac tgtaaaagtt gttgatgaat tggtcaaagt
     5641 aatggggcgg cataagccag aaaatatcgt tattgaaatg gcacgtgaaa atcagacaac
     5701 tcaaaagggc cagaaaaatt cgcgagagcg tatgaaacga atcgaagaag gtatcaaaga
     5761 attaggaagt cagattctta aagagcatcc tgttgaaaat actcaattgc aaaatgaaaa
     5821 gctctatctc tattatctcc aaaatggaag agacatgtat gtggaccaag aattagatat
     5881 taatcgttta agtgattatg atgtcgatca cattgttcca caaagtttcc ttgccgacga
     5941 ttcaatagac aataaggtct taacgcgttc tgataaaaat cgtggtaaat cggataacgt
     6001 tccaagtgaa gaagtagtca aaaagatgaa aaactattgg agacaacttc taaacgccaa
     6061 gttaatcact caacgtaagt ttgataattt aacgaaagct gaacgtggag gtttgagtga
     6121 acttgataaa gctggtttta tcaaacgcca attggttgaa actcgccaaa tcactaagca
     6181 tgtggcacaa attttggata gtcgcatgaa tactaaatac gatgaaaatg ataaacttat
     6241 tcgagaggtt aaagtgatta ccttaaaatc taaattagtt tctgacttcc gaaaagattt
     6301 ccaattctat aaagtacgtg agattaacaa ttaccatcat gcccatgatg cgtatctaaa
     6361 tgccgtcgtt ggaactgctt tgattaagaa atatccagcg cttgaatcgg agtttgtcta
     6421 tggtgattat aaagtttatg atgttcgtaa aatgattgct aagtctgagc aagaaatagg
     6481 caaagcaacc gcaaaatatt tcttttactc taatatcatg aacttcttca aaacagaaat
     6541 tacacttgca aatggagaga ttcgcaaagc ccctctaatc gaaactaatg gggaaactgg
     6601 agaaattgtc tgggataaag ggcgagattt tgccacagtg cgcaaagtat tgtccatgcc
     6661 ccaagtcaat attgtcaaga aaacagaagt acagacaggc ggattctcca aggagtcaat
     6721 tttaccaaaa agaaattcgg acaagcttat tgctcgtaaa aaagactggg atccaaaaaa
     6781 atatggtggt tttgatagtc caacggtagc ttattcagtc ctagtggttg ctaaggtgga
     6841 aaaagggaaa tcgaagaagt taaaatccgt taaagagtta ctagggatca caattatgga
     6901 aagaagttcc tttgaaaaaa atccgattga ctttttagaa gctaaaggat ataaggaagt
     6961 taaaaaagac ttaatcatta aactacctaa atatagtctt tttgagttag aaaacggtcg
     7021 taaacggatg ctggctagtg ccggagaatt acaaaaagga aatgagctgg ctctgccaag
     7081 caaatatgtg aattttttat atttagctag tcattatgaa aagttgaagg gtagtccaga
     7141 agataacgaa caaaaacaat tgtttgtgga gcagcataag cattatttag atgagattat
     7201 tgagcaaatc agtgaatttt ctaagcgtgt tattttagca gatgccaatt tagataaagt
     7261 tcttagtgca tataacaaac atagagacaa accaatacgt gaacaagcag aaaatattat
     7321 tcatttattt acgttgacga atcttggagc tcccgctgct tttaaatatt ttgatacaac
     7381 aattgatcgt aaacgatata cgtctacaaa agaagtttta gatgccactc ttatccatca
     7441 atccatcact ggtctttatg aaacacgcat tgatttgagt cagctaggag gtgactaaac
     7501 tagt
//
