LOCUS       sequence-409749-        8186 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GeoCas9-mediated cleavage of genomic DNA in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8186)
  TITLE     van der Oost E. coli CRISPR plasmids
REFERENCE   2  (bases 1 to 8186)
  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"
COMMENT     Sequence Label: pGCas9_scr
FEATURES             Location/Qualifiers
     source          1..8186
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(85..187)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(579..596)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(713..1258)
                     /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(793..812)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    4651..4675
                     /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        complement(join(4682..4687,4688..4704,4705..4710))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(4873..4892)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5098..5175
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5176..6258
                     /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     complement(5195..5214)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        6319..6353
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     CDS             complement(join(7611..8186,1..84))
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     join(8185..8186,1..18)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 aactgactga aatgcctcaa aatgttcttt acgatgccat tgggatatat caacggtggt
       61 atatccagtg atttttttct ccattttagc ttccttagct cctgaaaatc tcgataactc
      121 aaaaaatacg cccggtagtg atcttatttc attatggtga aagttggaac ctcttacgtg
      181 ccgatcaacg tctcattttc gccaaaagtt ggcccagggc ttcccggtat caacagggac
      241 accaggattt atttattctg cgaagtgatc ttccgtcaca ggtatttatt cggcgcaaag
      301 tgcgtcgggt gatgctgcca acttactgat ttagtgtatg atggtgtttt tgaggtgctc
      361 cagtggcttc tgtttctatc agctgtccct cctgttcagc tactgacggg gtggtgcgta
      421 acggcaaaag caccgccgga catcagcgct agcggagtgt atactggctt actatgttgg
      481 cactgatgag ggtgtcagtg aagtgcttca tgtggcagga gaaaaaaggc tgcaccggtg
      541 cgtcagcaga atatgtgata caggatatat tccgcttcct cgctcactga ctcgctacgc
      601 tcggtcgttc gactgcggcg agcggaaatg gcttacgaac ggggcggaga tttcctggaa
      661 gatgccagga agatacttaa cagggaagtg agagggccgc ggcaaagccg tttttccata
      721 ggctccgccc ccctgacaag catcacgaaa tctgacgctc aaatcagtgg tggcgaaacc
      781 cgacaggact ataaagatac caggcgtttc cccctggcgg ctccctcgtg cgctctcctg
      841 ttcctgcctt tcggtttacc ggtgtcattc cgctgttatg gccgcgtttg tctcattcca
      901 cgcctgacac tcagttccgg gtaggcagtt cgctccaagc tggactgtat gcacgaaccc
      961 cccgttcagt ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggaa
     1021 agacatgcaa aagcaccact ggcagcagcc actggtaatt gatttagagg agttagtctt
     1081 gaagtcatgc gccggttaag gctaaactga aaggacaagt tttggtgact gcgctcctcc
     1141 aagccagtta cctcggttca aagagttggt agctcagaga accttcgaaa aaccgccctg
     1201 caaggcggtt ttttcgtttt cagagcaaga gattacgcgc agaccaaaac gatctcaaga
     1261 agatcatctt attaatcaga taaaatattt gtcgacagga ttaaacaaaa atggcccgct
     1321 tcataagcag gccattttgt ctgatttagt cacgagtaga ttgcagtgga cgaatggttt
     1381 tgccaggttt gctgtgtgcg gaggacgcca gaccgacacg tttctcgcca cgcactttat
     1441 agatgttacc cagaacatca acttgatatt tctcgaaacg tttcagggtg cgagagccaa
     1501 cgccacgcag agagaaacgg tggtcgtgag aaatcagttc caggccgccg tttgcggagt
     1561 cgatcgtttt atagtaaacg aacacgtctt tcacgttaat ttcttcgcct gcagcggttt
     1621 taacggtttt ttcacgcggc agttcgatgc ggatcaggtc gttcggatac aggctgaaac
     1681 gaaacgtgta atcttcggtc atttctttcc attcagagta cggcttgttc ggttcaatcg
     1741 ctttgttcgg cagaatgcct ttcatgatgt ccatggtata caccggtaca cagtagtatt
     1801 taccatcttt ttcgaacacg tccacgcgaa cgatgttgga attgtaggcc acggttttac
     1861 catcgttcag agggatcacc tggtttttcg tgtcgataat ttttacagta cggattaccg
     1921 ggcccggttc gccgtttttt ttcggtttgt acagaggctc ctggaatgcc tttttcgggt
     1981 cgttgttatg ctccagcaga cgctggcgga tggcttcata agtgcgtgga tcggattctt
     2041 taccgtacat tgggaagtgg ccggatgcat ccagtttaat ctcggacagt ttggttttta
     2101 ctacggtctg gattttaccg gagcgttcgt cgatgcccac gtaacggcgc agcgtttcct
     2161 ggtgcgcagc gccagtaaca gaacgtttcg gcatacggct gacaaacacc ggctgcagag
     2221 attccagttt ttggtcatcg tagttaccca ggttcagagc tttgatgctt tctttaggat
     2281 gtttggacag acgagcacgc agttcgtccg cgaagtgcgg ccacggctgc ggaaaatgcg
     2341 gttcggtttt tttggccagt tccttgttct gctcacgacg ctggtagaat gcggtcacct
     2401 tagcgatatc gcttggggtg gtgcacgcca caataacggc gtcaacagcg tggtgcagat
     2461 cgctttcctc acggttcttg ttgaactccc agcgagaacg caggtgggcg gtaacacggc
     2521 cgtttacggt ataaactttc tgtttatcgt cggattccgc aaatttcagg tgttcacgaa
     2581 tgaagtttgc gaagaagcgg ctgatgtagc gagtatcgtt caggttacgg tttttgaatt
     2641 cagtctcttc gttttcatcg tagtgcagac gcagcagacg gtcacgtttc tttttagaga
     2701 actgtttatt ggtcaggacg aaggtttcga actgttgcca acgctcagtg ccaacaccca
     2761 gatattcagc tgggatgcgg ttgcctttct cacggttttc gcgagtcaga accagaactt
     2821 tgttggtata agaatcatcc agagagcggg agtacgggat cacatgatct acttcaacgt
     2881 aacctggctc cagcagacgt tcgatctcga tcggctgcag agagtaagca cagcgaccgt
     2941 tctgttcaga ccacagcttg aacttcacaa tatcgtgacc agtagggttc agagtcagac
     3001 cgtattccat cagctggcgg atcgcggttt cgtttttttt acggttttca tcctgttcct
     3061 ttttagtttt acgacgctcg tcaaaagtct ggctcaggtc gcgcgccagt tcgatatgga
     3121 tggaaaccgg gctgccgtac tttttgatga tcgcgttgac tactttgcgc gcctgggtca
     3181 gtgcacgcat tactacaggg ttcgcaatcg gcgggatgtt cggcagcagc atagtttttt
     3241 gcttcttttt cggaccggtg aaggtgtagc ctgcacgttc acaagcggag gagtagactt
     3301 caccctgttc catatacggc aggatggaac gcagagcttt cagagacagg tgaccgaatt
     3361 tagtgaagga caggttcagc agttcttcaa tcagctcgtt atcgtaaact ttgttagcca
     3421 ggttaggcat acgtttgccg ttctgttcgt actcgttgcg caggtaagag tggatatccg
     3481 catcgtcttt aaacagggtc agcgcgtaac cgaaggtatc aaagtcgatc ggcaggaagc
     3541 tgctggattt acccttgccg taaactttat ctacagcttt acggatctgg tggtatgcat
     3601 ccagttccag gaaacgaatg ttttcgttct gcttacgaga ttcaccgcga tcgtaaacga
     3661 tgcccttgaa gtaggtgtcg tccggcagat gcagcagggt acgaatatcg tggtaggtaa
     3721 ttttgttttt ctggaatgct tgttcataca gcagacgacg ttcttcgtcg gtcaggccgc
     3781 gggcgccaga cggagaaatc agacgcagtt tgttgatatg ttcccacgcg ataaaagact
     3841 ggaaggtata ggtggctttc ggcgcacgtt tttccttcgg ttcgaaagta caaaagccta
     3901 cttttttttc gatatcatct ttagacgcca ccggacgctg agatgcccaa atggtaatat
     3961 attcgttttc gaattcttcg gtgcaagaca tgttgccgaa ttcgcgctgt ttggagaaaa
     4021 tcagacggat ttcacgttcc aggtcatcgc gtgcaatggt gttggtgtag ttttcgcctt
     4081 tgttacgctt atgcagtgcg aatttcgggt ctttaacaat catttcgccc acggtacggt
     4141 agctagacag aattgcacgg ttctcctcaa tatgtttcag catagtggag ttctctttat
     4201 tggagcgttc agatttacgg ttagatttga agccacgacg ctttgccaga tgcagcagga
     4261 cacgcgccag ttcgtcgttg ttcagcttac ggtccagggc ttctacacgc agctgccata
     4321 cgtcgatctc gtgtttttct tcgaacagtt tatccagttc ttctttcgtc aggatgcctt
     4381 cacgaataac caggcgacga atacgctcca gacggtgttt acgacgacgc aggcggcggc
     4441 gtgcgctacg tgccagacga cgcggcagag ccagagattc accggtctgc gggttctccg
     4501 cacggtcaaa aatgcgcacg cccagatctt cgatacgagg gatatccagg ttcatgactg
     4561 cccaaccaac agaggtaata ccgatgtcca ggccaatctt ataacgcata cacggtgcct
     4621 gactgcgtta gcaatttaac tgtgataaac ggaattgtta tccgctcaca attcctaccg
     4681 cattaaagct tatcgatgat aagctgtcaa acatgagaat tacaacttat atcgtatggg
     4741 gctgacttca ggtgctacat ttgaagagat aaattgcact gaaatctaga tttcgcggga
     4801 tcgagatctc gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg
     4861 cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg
     4921 ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt
     4981 tgggcgccat ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc
     5041 tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga gatcccggac
     5101 accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa
     5161 ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc
     5221 tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg
     5281 gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa
     5341 ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg
     5401 ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg
     5461 gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc
     5521 gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct
     5581 gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc
     5641 atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc
     5701 gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt
     5761 ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa
     5821 cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag
     5881 ggtatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc
     5941 gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat
     6001 accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg
     6061 ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc
     6121 aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa
     6181 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     6241 ctggaaagcg ggcagtgatc agaatcgact gatggaccaa aaaaccccgc cgaagcgggg
     6301 actttgaact agcatacatt gacagctagc tcagtcctag gtataatgct agcctagatc
     6361 cgcagtaacc ccatgggtca tagttcccct gagattatcg ctgtggtata atgaaagtta
     6421 taccacagca atgatctcag ggttactatg ataagggctt tctgcctata ggcagactga
     6481 cccgtggcgt tggggatcgc ctatcgcccg ctttcttcgg gcattcccca ctcttaggcg
     6541 aacgaagacc ttggcggaaa acgtcaaggt cttttttatg gaagccggcg gcacctcgct
     6601 aacggattca ccactccaag aattggagcc aatcaattct tgcggagaac tgtgaatgcg
     6661 caaaccaacc cttggcagaa catatccatc gcgtccgcca tctccagcag ccgcacgcgg
     6721 cgcatctcgg gcagcgttgg gtcctggcca cgggtgcgca tgatcgtgct cctgtcgttg
     6781 aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc accgatacgc
     6841 gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg
     6901 gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcccctac gtgctgctga
     6961 agttgcccgc aacagagagt ggaaccaacc ggtgatacca cgatactatg actgagagtc
     7021 aacgccatga gcggcctcat ttcttattct gagttacaac agtccgcacc gctgccggta
     7081 gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta
     7141 tcatgcaact cgtaggacag gtgccggcag cgcccaacag tcccccggcc acggggcctg
     7201 ccaccatacc cacgccgaaa caagcgccct gcaccattat gttccggatc tgcatcgcag
     7261 gatgctgctg gctaccctgt ggaacaccta catctgtatt aacgaagcgc taaccgtttt
     7321 tatcaggctc tgggaggcag aataaatgat catatcgtca attattacct ccacggggag
     7381 agcctgagca aactggcctc aggcatttga gaagcacacg gtcacactgc ttccggtagt
     7441 caataaaccg gtaaaccagc aatagacata agcggctatt taacgaccct gccctgaacc
     7501 gacgaccggg tcgaatttgc tttcgaattt ctgccattca tccgcttatt atcacttatt
     7561 caggcgtagc aaccaggcgt ttaagggcac caataactgc cttaaaaaaa ttacgccccg
     7621 ccctgccact catcgcagta ctgttgtaat tcattaagca ttctgccgac atggaagcca
     7681 tcacaaacgg catgatgaac ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta
     7741 taatatttgc ccatggtgaa aacgggggcg aagaagttgt ccatattggc cacgtttaaa
     7801 tcaaaactgg tgaaactcac ccagggattg gctgagacga aaaacatatt ctcaataaac
     7861 cctttaggga aataggccag gttttcaccg taacacgcca catcttgcga atatatgtgt
     7921 agaaactgcc ggaaatcgtc gtggtattca ctccagagcg atgaaaacgt ttcagtttgc
     7981 tcatggaaaa cggtgtaaca agggtgaaca ctatcccata tcaccagctc accgtctttc
     8041 attgccatac ggaattccgg atgagcattc atcaggcggg caagaatgtg aataaaggcc
     8101 ggataaaact tgtgcttatt tttctttacg gtctttaaaa aggccgtaat atccagctga
     8161 acggtctggt tataggtaca ttgagc
//
