LOCUS       sequence-415374-        9308 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ga0116245_10002768 T7/LacO-Cas6 + TwinStrep-Cas14 + Cas7 + 
            His14-Cas5::pJ23119-DR-Fn spacer-DR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9308)
  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 9308)
  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: pHS1950
FEATURES             Location/Qualifiers
     source          1..9308
                     /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(4736..4770)
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     primer_bind     4981..5000
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5088..5107
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5088..5106
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5107..5131
                     /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             5146..5168
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(5879..5902,5903..5938,5939..5962)
                     /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      9236..9283
                     /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 gtggcgataa 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 gttttaaccc caacatatgg gacttaaacg agaagtcatt
     4681 taataaggcc actgttaaaa agcttagttt taaccccacc atatgggact taaacgctag
     4741 tattatacct aggactgagc tagctgtcaa cattaattcc taatttttct ctcccttatg
     4801 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     4861 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     4921 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     4981 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     5041 gatgcgtccg gcgtagagga tcgagatcga tctcgatccc gcgaaattaa tacgactcac
     5101 tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa
     5161 gaaggagata taccatgaac gagatgcggg tagaaatttc ttttgggacc atagaaaaac
     5221 caccaattca ttcaatccaa tcatttatat atcaatgcct tcatgaggaa gataaagaat
     5281 atgctacatt tatacacaaa gagggcatca aacataagtg gaaatcaatc aagccattta
     5341 tcttttccca cccgtacatt aagaaggata acaagtgtta tatcaaagtt agcagcctgg
     5401 acagcttttt caactataac ttcttctctg gtttaaataa gatcatgttc aaaaagaaca
     5461 acgctatcat taagccggaa tcgattcgta ttatcaatat tccgaagatt aagtacgata
     5521 gcaatggcaa cacccagcaa aacatgttct tcatctcccc gctgctggtg aagtccaata
     5581 gcggttacat caaagacccg gacgacgaaa aattccttca gatcttgaag aagaatctga
     5641 tcgataaata cctggcgatt aacaaaaagg agtgcaccaa cgatacgttt tctatttttc
     5701 tgaaagacaa aaccccgaaa ttgaaaattt ataagaaaga ggaattgccg gtttttctga
     5761 gcaaattcgt gctgatttcg agcaaagaac tttttaatat tgcttattat ctcggactcg
     5821 ggtgtaagaa tgcactcggt tttggaatga ttgagtttga tagggaggat ttatgatatg
     5881 gagccatccg cagtttgaaa aaggtggtgg tagcggtggt ggttcaggtg gtagtgcatg
     5941 gtcacaccct cagtttgaga aatcaagtgg atccatgata aaatttattg gtggagcatc
     6001 aaaagttact ggttcagcat ttttacttga gactggaaat gcaaaaattt taattgattg
     6061 tggtattgaa caagaaaagg gaatagagaa agataacaac gaaatcatcg aaaagaagat
     6121 taacgagatc ggcaaggccg atatctgcat tcttactcac gcgcatctgg accacagcgg
     6181 cctggtgccg ctgctggtga aaaagcgcaa agtaaataag atcatcagca cgcctgcgac
     6241 caaagagctt tgccgtttgt tgttcaacga cttccagcgt attcaagaag agaataacga
     6301 cattccgctg tacagctatg atgacatcga gagcagcttt gaaatttggg atgaaattga
     6361 cgatcgtaat accattgagt tgttcgacac caaaatcacc ttttataaca actctcacat
     6421 tattggctca gttagcgtgt tcatcgaaac ccacaatgga aattacctgt tctcgggcga
     6481 cattggctcc aaactacaac agctgatgga ttatccgccg gacatgccgg atggtaatgt
     6541 tgactacttg atcctagaga gcacctatgg taacaaaagc cacgactcta gcgatcgtga
     6601 tcgattgctg gaaatagcga aaaccacgtg cgaaaacggc ggcaaagtgc tgattccgag
     6661 cttcgccatc ggcagattgc aagaagtgct ctacaccttt tctaactata actttaactt
     6721 cccggtttat atcgatagcc cgatgggttc aaaggtcacc aatctgatca aagagtacaa
     6781 catttatctg aaaaagaaac tccgtcgcct gagcattact gacgacctgt tcaataacaa
     6841 gtacatcgcc attaatacga gcaaccagtc taaagagctg tctaacagca aggaaccggc
     6901 ggttattatt agcgcgagcg ggatgctgga gggtggccgc attctgaatc atttggagca
     6961 gatcaagaat gacgaaaact ccaccctgat ctttgttggt tatcaggcac agaacacccg
     7021 tggtcgcaag atcttggacg gcgaagagaa agtgcgttgt cgtatagaga agctgaatag
     7081 ctttagtgcg catgctgatc aggatgagct gatcgactac attgaacgct taaagtacac
     7141 cccgtataaa gtgttcctgg tgcatggtga aaaggagcaa cgtgaaattt tagcgaaacg
     7201 tatcatcagc aagaaaatcc gtgttgagct gccagaaaac tactcccaag gtaaagaaat
     7261 cttgatcgag aagaaggtcg tcctgaacat taatactgac aatatgtgta attttgcttc
     7321 ctaccgcctg atgccgtttt cgggttttat cgtggaaaag gacgaccgca tcgaaataaa
     7381 cgataaaaac tggttcgata tgatttggaa tgaggagtac aataagatgc gtagccaaat
     7441 tgttgctgaa gatttctcca ccgaccaaaa cgaggacagc atggcgctgc cggatatgag
     7501 ccacgataag attattgaga acattgaata cctcttcaat atcaaaatcc tctcgaagaa
     7561 ccgtatcaaa gagttttggg aagagttttg caaaggtcag aaagcggcaa tcaagtacat
     7621 tacccaggtt catcgtaaga acccgaatac gggtcgtcgc aactggaacc cgccagaggg
     7681 tgatttcacg gacaacgaga tcgagaagct gtatgaaact gcatataata cattgcttag
     7741 tttaataaaa tatgataaaa ataaagtcta taatatttta ataaatttta atccaaaatt
     7801 ataaaaatta taaaaggagt ttttatggca aaaacaatga aaaaaattta tgtaacaatg
     7861 aagactttat cacctcttta cactggtgag gtaagaagag aggataagga agctgctcaa
     7921 aaaagagtaa attttcccgt tcgtaagacc gcaacgaata aagttctgat cccgtttaag
     7981 ggcgcgttgc gtagcgcgtt ggaaatcatg ctgaaagcta aaggcgaaaa cgtgtgcgac
     8041 actggtgagt cccgtgcccg tccgtgtggt cgctgcgtca cgtgcagcct gtttggtagc
     8101 atgggtcgcg cgggtcgcgc gagcgttgat tttctgatct cgaatgacac caaagaacag
     8161 attgtgcgtg aatccaccca cctgcgtatt gagagacaga ccaagtctgc cagcgatacc
     8221 ttcaagggcg aggaagttat tgagggtgca acgttcaccg cgaccattac catctctaac
     8281 ccgcaagaga aagacctgag ccttatccaa agcgctctca agttcatcga agagaacgga
     8341 ataggaggct ggcttaataa aggttatggt agagtaagtt ttgaagttaa atcagaagat
     8401 gtggcaacag atagttctta aaataggaag tattaaaatg agcaagcatc accatcattc
     8461 aggccatcac cataccggac accaccatca ttcaggcagt catcaccata ccatgaaaga
     8521 gataaaagga attttagaat ctattactgg ttttagcatc cctcttgata atggtgaata
     8581 tgctctttat ccagcaggta gacacctaag gggtgcaatt ggatatatag ctttcaacct
     8641 ggatctgcca atctcctcta agttcctgga ctttgatttc gacgacatca tcttccgcga
     8701 tctgctgccg attagcaaat gtggtaagat cttttatccg gaaaagaaca gcaactcgct
     8761 gaaatgcccg agctgcaacg agatctacgg cagcagtgta ctgcgtaata ttatggctag
     8821 aggcctgagc tacaaggagg tgattgaagg taaaaagtac cgcttgtcta ttatcgtgaa
     8881 ggacgaaaaa tacttgaacg agatggaagc gatcattcgt tatattctgt cctacggcat
     8941 ttacctgggt aataaagtta gcaagggtta tggcaaattt aaaatcaagg agtatagcat
     9001 tgtggatatc ctcccggtta aagacagcga agttttgtta ttgtcggatg caattatcga
     9061 caatggtgaa aaggacatcg tctttagcaa gaaggagatt tcctcttcca aattcgagat
     9121 catccgtaaa cgtggcaagg cgaaagggga tataataaga gacaataatc ataatggttt
     9181 ttatatcggt aagtatggtg gacttggctt tggagagata atttccttaa aatgactagc
     9241 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
     9301 atccggat
//
