LOCUS       sequence-416238-        9259 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ga0116245_10002768 T7/LacO-Cas6 + Cas14-TwinStrep + Cas7 + 
            His14-Cas5::pJ23119-truncated DR-truncated Fn spacer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9259)
  AUTHORS   Altae-Tran H, Kannan S, Suberski AJ, Mears KS, Demircioglu FE, 
            Moeller L, Kocalar S, Oshiro R, Makarova KS, Macrae RK, Koonin EV, 
            Zhang F
  TITLE     Uncovering the functional diversity of rare CRISPR-Cas systems with 
            deep terascale clustering.
  JOURNAL   Science. 2023 Nov 24;382(6673):eadi1910. doi: 
            10.1126/science.adi1910. Epub 2023 Nov 23.
  PUBMED    37995242
REFERENCE   2  (bases 1 to 9259)
  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: "Science."; 
            date: "2023-11-24"; volume: "382(6673):eadi1910. doi"; pages: " 
            10.1126/science.adi1910. Epub 2023 Nov 23"
COMMENT     Sequence Label: pCX0393
FEATURES             Location/Qualifiers
     source          1..9259
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     309..330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(582..1373,1374..1442))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGRPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1205..1224
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1443..1535)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1616..2204
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2105..2124
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2358..2375
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2390..2529
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2455..2474)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2615..2637
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2631..2822)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3394..4476)
                     /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     4438..4457
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4477..4554)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      complement(4568..4639)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        complement(4693..4727)
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     primer_bind     4938..4957
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5045..5064
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5045..5063
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5064..5088
                     /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             5103..5125
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(7669..7692,7693..7728,7729..7752)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     terminator      9187..9234
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttctg gcggcacgat
      481 ggcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
      541 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
      601 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
      661 gtgtagataa ctacgatacg ggagggccta ccatctggcc ccagtgctgc aatgataccg
      721 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
      781 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
      841 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
      901 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
      961 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     1021 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     1081 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     1141 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     1201 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     1261 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     1321 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     1381 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     1441 atactcttcc tttttcaatc atgattgaag catttatcag ggttattgtc tcatgagcgg
     1501 atacatattt gaatgtattt agaaaaataa acaaataggt catgaccaaa atcccttaac
     1561 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     1621 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     1681 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     1741 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     1801 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     1861 gtggtgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     1921 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     1981 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     2041 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     2101 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2161 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2221 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     2281 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     2341 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     2401 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     2461 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     2521 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     2581 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     2641 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     2701 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     2761 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     2821 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     2881 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     2941 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     3001 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     3061 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     3121 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     3181 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     3241 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc tagtcatgcc ccgcgcccac
     3301 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     3361 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3421 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3481 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     3541 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     3601 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     3661 tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     3721 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     3781 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     3841 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     3901 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     3961 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     4021 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     4081 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     4141 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     4201 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     4261 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     4321 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     4381 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     4441 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     4501 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     4561 ctcgacggag agcgttcacc gacaaacaac agataaaacg aaaggcccag tctttcgact
     4621 gagcctttcg ttttatttga taaggccact gttaaaaagc ttagttttaa ccccaccata
     4681 tgggacttaa acgctagtat tatacctagg actgagctag ctgtcaacat taattcctaa
     4741 tttttctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     4801 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     4861 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     4921 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     4981 ccggtgatgc cggccacgat gcatccggcg tagaggatcg agatcgatct cgatcccgcg
     5041 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     5101 attttgttta actttaagaa ggagatatac catgaacgag atgcgggtag aaatttcttt
     5161 tgggaccata gaaaaaccac caattcattc aatccaatca tttatatatc aatgccttca
     5221 tgaggaagat aaagaatatg ctacatttat acacaaagag ggcatcaaac ataagtggaa
     5281 atcaatcaag ccatttatct tttcccaccc gtacattaag aaggataaca agtgttatat
     5341 caaagttagc agcctggaca gctttttcaa ctataacttc ttctctggtt taaataagat
     5401 catgttcaaa aagaacaacg ctatcattaa gccggaatcg attcgtatta tcaatattcc
     5461 gaagattaag tacgatagca atggcaacac ccagcaaaac atgttcttca tctccccgct
     5521 gctggtgaag tccaatagcg gttacatcaa agacccggac gacgaaaaat tccttcagat
     5581 cttgaagaag aatctgatcg ataaatacct ggcgattaac aaaaaggagt gcaccaacga
     5641 tacgttttct atttttctga aagacaaaac cccggaattg aaaatttata agaaagagga
     5701 attgccggtt tttctgagca aattcgtgct gatttcgagc aaagaacttt ttaatattgc
     5761 ttattatctc ggactcgggt gtaagaatgc actcggtttt ggaatgattg agttcgatag
     5821 ggaggattta tgataaaatt tattggtgga gcatcaaaag ttactggttc agcattttta
     5881 cttgagactg gaaatgcaaa aattttaatt gattgtggta ttgaacaaga aaagggaata
     5941 gagaaagata acaacgaaat catcgaaaag aagattaacg agatcggcaa ggccgatatc
     6001 tgcattctta ctcacgcgca tctggaccac agcggcctgg tgccgctgct ggtgaaaaag
     6061 cgcaaagtaa ataagatcat cagcacgcct gcgaccaaag agctttgccg tttgttgttc
     6121 aacgacttcc agcgtattca agaagagaat aacgacattc cgctgtacag ctatgatgac
     6181 atcgagagca gctttgaaat ttgggatgaa attgacgatc gtaataccat tgagttgttc
     6241 gacaccaaaa tcacctttta taacaactct cacattattg gctcagttag cgtgttcatc
     6301 gaaacccaca atggaaatta cctgttctcg ggcgacattg gctccaaact acaacagctg
     6361 atggattatc cgccggacat gccggatggt aatgttgact acttgatcct agagagcacc
     6421 tatggtaaca aaagccacga ctctagcgat cgtgatcgat tgctggaaat agcgaaaacc
     6481 acgtgcgaaa acggcggcaa agtgctgatt ccgagcttcg ccatcggcag attgcaagaa
     6541 gtgctctaca ccttttctaa ctataacttt aacttcccgg tttatatcga tagcccgatg
     6601 ggttcaaagg tcaccaatct gatcaaagag tacaacattt atctgaaaaa gaaactccgt
     6661 cgcctgagca ttactgacga cctgttcaat aacaagtaca tcgccattaa tacgagcaac
     6721 cagtctaaag agctgtctaa cagcaaggaa ccggcggtta ttattagcgc gagcgggatg
     6781 ctggagggtg gccgcattct gaatcatttg gagcagatca agaatgacga aaactccacc
     6841 ctgatctttg ttggttatca ggcacagaac acccgtggtc gcaagatctt ggacggcgaa
     6901 gagaaagtgc gttgtcgtat agagaagctg aatagcttta gtgcgcatgc tgatcaggat
     6961 gagctgatcg actacattga acgcttaaag tacaccccgt ataaagtgtt cctggtgcat
     7021 ggtgaaaagg agcaacgtga aattttagcg aaacgtatca tcagcaagaa aatccgtgtt
     7081 gagctgccag aaaactactc ccaaggtaaa gaaatcttga tcgagaagaa ggtcgtcctg
     7141 aacattaata ctgacaatat gtgtaatttt gcttcctacc gcctgatgcc gttttcgggt
     7201 tttatcgtgg aaaaggacga ccgcatcgaa ataaacgata aaaactggtt cgatatgatt
     7261 tggaatgagg agtacaataa gatgcgtagc caaattgttg ctgaagattt ctccaccgac
     7321 caaaacgagg acagcatggc gctgccggat atgagccacg ataagattat tgagaacatt
     7381 gaatacctct tcaatatcaa aatcctctcg aagaaccgta tcaaagagtt ttgggaagag
     7441 ttttgcaaag gtcagaaagc ggcaatcaag tacattaccc aggttcatcg taagaacccg
     7501 aatacgggtc gtcgcaactg gaacccgcca gagggtgatt tcacggacaa cgagatcgag
     7561 aagctgtatg aaactgcata taatacattg cttagtttaa taaaatatga taaaaataaa
     7621 gtctataata ttttaataaa ttttaatcca aaattaggca gcgctagctg gagccatccg
     7681 cagtttgaaa aaggtggtgg tagcggtggt ggttcaggtg gtagtgcatg gtcacaccct
     7741 cagtttgaga aataaaaatt ataaaaggag tttttatggc aaaaacaatg aaaaaaattt
     7801 atgtaacaat gaagacttta tcacctcttt acactggtga ggtaagaaga gaggataagg
     7861 aagctgctca aaaaagagta aattttcccg ttcgtaagac cgcaacgaat aaagttctga
     7921 tcccgtttaa gggcgcgttg cgtagcgcgt tggaaatcat gctgaaagct aaaggcgaaa
     7981 acgtgtgcga cactggtgag tcccgtgccc gtccgtgtgg tcgctgcgtc acgtgcagcc
     8041 tgtttggtag catgggtcgc gcgggtcgcg cgagcgttga ttttctgatc tcgaatgaca
     8101 ccaaagaaca gattgtgcgt gaatccaccc acctgcgtat tgagagacag accaagtctg
     8161 ccagcgatac cttcaagggc gaggaagtta ttgagggtgc aacgttcacc gcgaccatta
     8221 ccatctctaa cccgcaagag aaagacctga gccttatcca aagcgctctc aagttcatcg
     8281 aagagaacgg aataggaggc tggcttaata aaggttatgg tagagtaagt tttgaagtta
     8341 aatcagaaga tgtggcaaca gatagttctt aaaataggaa gtattaaaat gagcaagcat
     8401 caccatcatt caggccatca ccataccgga caccaccatc attcaggcag tcatcaccat
     8461 accatgaaag agataaaagg aattttagaa tctattactg gttttagcat ccctcttgat
     8521 aatggtgaat atgctcttta tccagcaggt agacacctaa ggggtgcaat tggatatata
     8581 gctttcaacc tggatctgcc aatctcctct aagttcctgg actttgattt cgacgacatc
     8641 atcttccgcg atctgctgcc gattagcaaa tgtggtaaga tcttttatcc ggaaaagaac
     8701 agcaactcgc tgaaatgccc gagctgcaac gagatctacg gcagcagtgt actgcgtaat
     8761 attatggcta gaggcctgag ctacaaggag gtgattgaag gtaaaaagta ccgcttgtct
     8821 attatcgtga aggacgaaaa atacttgaac gagatggaag cgatcattcg ttatattctg
     8881 tcctacggca tttacctggg taataaagtt agcaagggtt atggcaaatt taaaatcaag
     8941 gagtatagca ttgtggatat cctcccggtt aaagacagcg aagttttgtt attgtcggat
     9001 gcaattatcg acaatggtga aaaggacatc gtctttagca agaaggagat ttcctcttcc
     9061 aaattcgaga tcatccgtaa acgtggcaag gcgaaagggg atataataag agacaataat
     9121 cataatggtt tttatatcgg taagtatggt ggacttggct ttggagagat aatttcctta
     9181 aaatgactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     9241 ggaggaacta tatccggat
//
