LOCUS       sequence-426090-        8969 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GG entry for zCas9i expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8969)
  AUTHORS   Piepers M, Erbstein K, Reyes-Hernandez J, Song C, Tessi T, 
            Petrasiunaite V, Faerber N, Distel K, Maizel A
  TITLE     GreenGate 2.0: Backwards compatible addons for assembly of complex 
            transcriptional units and their stacking with GreenGate.
  JOURNAL   PLoS One. 2023 Sep 8;18(9):e0290097. doi: 
            10.1371/journal.pone.0290097. eCollection 2023.
  PUBMED    37682951
REFERENCE   2  (bases 1 to 8969)
  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"; journalName: "PLoS One.";
            date: "2023-09-8"; volume: "18(9):e0290097. doi"; pages: " 
            10.1371/journal.pone.0290097. eCollection 2023"
COMMENT     Sequence Label: pKE014
FEATURES             Location/Qualifiers
     source          1..8969
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(201..269,270..1061)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(419..438)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1721..1740
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1974..1991
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2108..2129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2144..2149,2150..2167,2168..2174)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /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        2212..2230
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2212..2229
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             2267..2287
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8020..8040
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(8555..8574)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8556..8574)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8584..8606)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(8800..8819)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8919..8941
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtggctct cgaggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacgagc tatttaggtg
     2221 acactataga agtgaagctt ggtctcaaac aatggcttct agcccaccga agaagaagcg
     2281 gaaggtcagc tggaaaatgg acaagaagta cagcattgga cttgatattg gtacgaactc
     2341 agttgggtgg gccgttatca ccgatgaata caaggtacct tcgaagaaat ttaaagtgct
     2401 gggcaacaca gataggcaca gcattaagaa gaacttgatc ggagctctgc tctttgactc
     2461 tggagaaacc gcggaggcga caaggcttaa acgtactgcg aggagaaggt acactcgcag
     2521 gaagaacaga atctgttatc tccaagagat ctttagcaac gagatggcga aggtaaggat
     2581 ttttatgata tagtatgctt atgtattttg tactgaaagc atatcctgct tcattgggat
     2641 attactgaaa gcatttaact acatgtaaac tcacttgatg atcaataaac ttgattttgc
     2701 aggttgacga ctcgttcttc catcgcctcg aggaatcctt cctggtagag gaagataaga
     2761 aacacgagcg tcaccccatc tttgggaata ttgttgacga agtagcctat catgaaaagt
     2821 atccgactat ataccacctt cgcaagaagc tggtggactc aaccgataag gcagaccttc
     2881 ggctcatata cctggctctc gcgcacatga taaagtttcg tggccatttc ttgatcgaag
     2941 gggacctcaa cccggataac tccgatgtgg ataaactgtt cattcagctc gtccaaacct
     3001 acaatcagct gttcgaggag aaccccatca atgcatcagg taacattcct tagttacctt
     3061 tcttttcttt ttccatcata agtttataga ttgtacatgc tttgagattt ttctttgcaa
     3121 acaatctcag gtgtcgacgc caaggcaata ctgtctgcca gactttcgaa gtccagacgg
     3181 cttgagaatc tgatcgctca attgccaggc gagaagaaga acggcttgtt cgggaatctg
     3241 attgcactgt ctctgggcct cacccctaac ttcaaaagca actttgacct cgccgaggac
     3301 gcgaagctgc agctgtcaaa ggatacatac gatgatgatc tggacaatct gctcgcccaa
     3361 ataggtgctc ttgaaattgg aactcttctt ttgttgtcta aacctatcaa tttctttgcg
     3421 gaaatttatt tgaagctgta gagttaaaat tgagtctttt aaacttttgt aggtgatcag
     3481 tatgccgacc tgttcttggc tgccaagaat ctgtcagacg ctatcttgct cagtgacatt
     3541 ctgcgggtca acacggagat aaccaaagcg ccacttagcg cctccatgat caagaggtac
     3601 gacgagcatc accaggatct gacccttctg aaggctttgg ttcgccagca actccccgag
     3661 aagtacaagg agattttctt tgaccaatcg aagaatggct acgcagggta cattgatgga
     3721 ggtaagttgt tacttatgat tgttttcctc tctgctacat gtattttgtt gttcatttct
     3781 gtaagatata agaattgagt tttcctctga tgatattatt aggtgcaagt caggaggaat
     3841 tctacaaatt catcaagcct attctggaaa agatggacgg tacagaggag ctgctcgtta
     3901 aattgaaccg cgaagatttg cttcggaagc agcgtacctt cgacaatggc agcataccgc
     3961 accagatcca cctcggtgag ctgcatgcta tcttgaggag gcaagaggac ttctatccgt
     4021 tcctgaaaga caacagagag aagattgaaa agatcctcac gttccgcatt ccctactatg
     4081 taggttagta tcatatgaag aaatacctag tttcagttga tgaatgctat tttctgacct
     4141 cagttgttct cttttgagaa ttatttcttt tctaatttgc ctgatttttc tattaattca
     4201 ttaggtccac tcgcacgcgg gaactcgcgg tttgcgtgga tgacacgcaa atccgaggag
     4261 actatcacgc cttggaactt cgaagaggtc gtggacaagg gtgcgagtgc acagtccttc
     4321 atcgaaagga tgaccaactt cgataagaat ctcccaaatg agaaagtcct gcccaagcat
     4381 agtctcctgt acgaatactt cacggtctac aacgagctga cgaaggtgaa atatgtgacg
     4441 gaggggatgc gcaaaccggc cttcctgtca ggtaaatcct ggtccacact tttacgataa
     4501 aaacacaaga ttttaaacta tgaactgatc aataatcatt cctaaaagac cacacttttg
     4561 ttttgtttct aaagtaattt ttactgttat aacaggtgag cagaagaagg ccattgtcga
     4621 tctcttgttc aaaaccaatc ggaaggtcac tgtgaaacag cttaaagagg actactttaa
     4681 gaagatcgaa tgctttgatt ctgtggaaat cagcggcgtt gaggataggt tcaatgcctc
     4741 tcttggcaca taccatgacc tgttgaaaat catcaaggac aaggacttcc ttgacaacga
     4801 ggagaacgag gacatcctcg aggacatcgt gctgactctc acgctgtttg aggacagaga
     4861 aatgatcgag gagcgcctta agacttatgc gcatctgttc gatgacaagg tcatgaagca
     4921 gttgaagagg aggagatata caggtaagag gtcaaaaggt ttccgcaatg atccctcttt
     4981 ttttgtttct ctagtttcaa gaatttgggt atatgactaa cttctgagtg ttccttgatg
     5041 catatttgtg atgagacaaa tgtttgttct atgttttagg ttggggaagg ctctccagga
     5101 agctcatcaa cggcatccgc gacaagcaat ccggcaagac tatactggac tttctcaaat
     5161 ccgacggttt tgcgaatcgg aacttcatgc agcttattca cgatgactca ctgaccttca
     5221 aagaagatat ccagaaggcc caagtgtcag gtcagggcga tagccttcac gaacacatag
     5281 ccaacctggc tggatcgcca gctataaaga agggcatact gcagacagtg aaggttgtgg
     5341 atgagctggt gaaggtaagt tctgcatttg gttatgctcc ttgcatttta ggtgttcgtc
     5401 gcacttccat ttccatgaat agctaagatt ttttttctct gcattcattc ttcttgcctc
     5461 agttctaact gtttgtggta tttttgtttt aattattgct acaggtcatg ggccgccata
     5521 agccggagaa catcgtcatc gagatggcga gggaaaacca gacgactcag aaagggcaga
     5581 agaactcacg ggagcgcatg aagcggatag aggaaggcat caaggagctt gggagtcaga
     5641 ttctgaaaga gcacccagtc gaaaatactc aactccagaa cgagaagctg tacctctatt
     5701 acctccagaa tgggagagat atgtacgtcg accaagagct cgacattaac agactctccg
     5761 actatgatgt ggatcacatt gtccctcaat ctttcctgaa ggacgatagt attgacaaca
     5821 aggtaaagca actgtgtttt aatcaatttc ttgtcaggat atatggatta taacttaatt
     5881 tttgagaaat ctgtagtatt tggcgtgaaa tgagtttgct ttttggtttc tcccgtgtta
     5941 taggtcctta cgcgctcaga caagaaccgc ggaaaatccg acaatgtacc cagcgaggag
     6001 gttgtgaaga agatgaagaa ctattggagg cagcttttga atgctaagct cataacccaa
     6061 cggaaattcg acaatctcac gaaggcagaa aggggcggac tgtctgagct cgacaaagcc
     6121 ggcttcatca agcgccagtt ggttgaaact cgtcagatta cgaaacatgt ggcccagata
     6181 ctcgattcgc gtatgaatac gaagtatgat gagaatgaca aacttatcag ggaggtaaag
     6241 gtaaagtttc caactttcct ttaccatatc aaactaaagt tcgaaacttt ttatttgatc
     6301 aacttcaagg ccacccgatc tttctattcc tgattaattt gtgatgaatc catattgact
     6361 tttgatggtt acgcaggtga tcaccctcaa gagcaaactg gttagtgact tccggaagga
     6421 cttccagttt tacaaggttc gcgagatcaa caactaccat catgcccatg acgcctacct
     6481 gaacgccgtt gttggcactg ctctcatcaa gaagtatccg aaactggagt ctgagtttgt
     6541 gtacggggat tacaaggtgt acgacgttag gaagatgatc gcgaagtcag aacaagagat
     6601 cggcaaggct accgcgaaat acttctttta ctcgaatatc atgaacttct tcaagacaga
     6661 gatcactctg gcgaatggtg aaatccggaa gaggcctctg atcgagacaa atggcgaaac
     6721 aggtctgtct ttcctatttc atatgtttaa tcctaggaat ttgatcaatt gattgtatgt
     6781 atgtcgatcc caagactttc ttgttcactt atatcttaac tctctctttg ctgtttcttg
     6841 caggtgagat tgtctgggat aagggcaggg attttgcgac tgtgcgtaag gttctcagca
     6901 tgccccaagt caacatagtc aagaaaacgg aggttcaaac cggtggtttc tccaaggagt
     6961 ccattctccc taagcgcaac tccgacaaac tgattgcgag gaagaaggat tgggatccga
     7021 agaaatacgg aggctttgat agccctaccg tggcatacag cgtactggta gtggccaagg
     7081 tggagaaggg caagagcaag aaactgaaaa gcgtcaagga actgcttgga attaccataa
     7141 tggaaaggtc ctcgttcgag aagaatccga tcgacttcct cgaggctaaa ggtaaaatat
     7201 tggatgccag acgatattct ttcttttgat ttgtaacttt ttcctgtcaa ggtcgataaa
     7261 ttttattttt tttggtaaaa ggtcgataat ttttttttgg agccattatg taattttcct
     7321 aattaactga accaaaatta tacaaaccag gttacaaaga ggtgaagaaa gacctcatta
     7381 tcaaactgcc caagtattcg cttttcgaat tggaaaatgg cagaaaacgc atgctggcat
     7441 ctgccggaga actgcagaag ggcaacgagc tggcattgcc cagtaagtac gtcaacttcc
     7501 tgtacttggc ctcacactat gagaagctga aggggtcacc agaggacaac gagcagaagc
     7561 agttgtttgt cgagcagcac aagcactatc ttgatgagat catagagcag atcagcgaat
     7621 tttccaagcg ggtcattctt gcagacgcta acctcgataa ggtaaggact tctcatgaat
     7681 attagtggca gattagtgtt gttaaagtct ttggttagat aatcgatgcc tcctaattgt
     7741 ccatgtttta ctggttttct acaattaaag gtgctttccg cgtacaacaa gcacagagat
     7801 aagccgataa gggaacaagc ggaaaacatc atccacctgt tcacactgac caatctggga
     7861 gccccagcag cctttaagta cttcgatacc actatcgaca gaaagcgcta cacatcaacc
     7921 aaggaagtgt tggacgctac ccttattcac caatctatta cagggctcta tgagacaagg
     7981 atagatctgt cgcagttggg tggtgactct agggctgacc caaagaagaa gcgtaaagtc
     8041 tgaggcgcgc cgagctccag gcctcccagc tttcgtccgt atcatcggtt tcgacaacgt
     8101 tcgtcaagtt caatgcatca gtttcattgc ccacacacca gaatcctact aagtttgagt
     8161 attatggcat tggaaaagct gttttcttct atcatttgtt ctgcttgtaa tttactgtgt
     8221 tctttcagtt tttgttttcg gacatcaaaa tgcaaatgga tggataagag ttaataaatg
     8281 atatggtcct tttgttcatt ctcaaattat tattatctgt tgtttttact ttaatgggtt
     8341 gaatttaagt aagaaaggaa ctaacagtgt gatattaagg tgcaatgtta gacatataaa
     8401 acagtctttc acctctcttt ggttatgtct tgaattggtt tgtttcttca cttatctgtg
     8461 taatcaagtt tactatgagt ctatgatcaa gtaattatgc aatcaagtta agtacagtat
     8521 aggcttgagc tcccggcttg agaccgaatt ctcgccctat agtgagtcgt attacctggc
     8581 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     8641 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     8701 ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca
     8761 tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc
     8821 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     8881 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     8941 gttttcaccg tcatcaccga aacgcgcga
//
