LOCUS       sequence-416243-        7057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FLAG-SV40 NLS-Cas8/Cse1 from MWEE01000013 Cas5-HNH in pcDNA3.1(+).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7057)
  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 7057)
  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: pHS1514
FEATURES             Location/Qualifiers
     source          1..7057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     612..631
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(850..954)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1029..1048)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1220..1599
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1600..1803
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1754..1774
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1848..1867
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1848..1866
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1902..1911
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1911..1934
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1947..1967
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3633..3650)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3639..3863
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3909..4337
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3996..4015)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4206..4227
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4346..4366)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4351..4680
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4531..4666
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4593..4612
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4747..5541
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4801..4820)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5411..5430
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5715..5836
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5752..5771)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5806..5825
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5885..5901)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5885..5901)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5898..5920)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5909..5925
                     /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(5933..5939,5940..5957,5958..5963))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5978..5999
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6116..6133)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6287..6875)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6367..6386)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7046..7057,1..780,781..849))
                     /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"
ORIGIN
        1 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
       61 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
      121 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
      181 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
      241 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
      301 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
      361 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
      421 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
      481 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
      541 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
      601 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
      661 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
      721 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
      781 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
      841 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
      901 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
      961 tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc
     1021 actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt
     1081 gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg
     1141 accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
     1201 cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg
     1261 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
     1321 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
     1381 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     1441 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
     1501 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
     1561 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
     1621 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
     1681 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
     1741 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
     1801 gctctctggc taactagaga acccactgct tactggctta tcgaaattaa tacgactcac
     1861 tatagggaga cccaagctgg ctagcgttta aacttaagct tgccaccatg gactataagg
     1921 atgatgacga caagatggcc ggatctccaa aaaagaaacg gaaggtggga tctatgtatt
     1981 gcgcggcggt ttgctttccg cagacaaaat atcagcaaag gggaactgct ctaaagaaac
     2041 cgctcgtcgg tctgcgtaag atgggtgtgg aagcggctgc atggaacacc ctgaaggtta
     2101 ctagagatcg ccctaagttg acctttccgg atctgatcac tccgcaaagc aaattcctgg
     2161 ataacgactt atggctgaag tacaaagtgc caattgagca gaaagaacac gtcatgtata
     2221 acctgttgtg cgataactgg gttaatgttg tttacttgag cggtaaaccg gaccgtattt
     2281 ccttagtcca aaccctgaag gacgcccatt gtctgcaatt ggcgtattct aacccgatgg
     2341 atcgtttcac cgtgttccgc ttcctgttgg cgttgggtta ctggtgtttc gcgaacacga
     2401 acgtcgagcc ggagcccgac aaaccgctgc cggtgagctg gattccgtgg ctggaggaaa
     2461 ataaagagta cttcgagctg ttcggtgatg gtaaacgttt cttccaagct gacccgagca
     2521 gccgtattcg tgcgattact gacctgatcc atgaaattcc gaccgctcac aacctgtgcc
     2581 attttaagca cgtgaccgac tatatcgacg gcctgtgtga agcctgctgc ataaaaggtc
     2641 tgttgcgtct gccggtattt accaccgttg gcggtcgtgg cattggcgca ggtatcaata
     2701 acacgccacc gttttatctg ctgtggcatg cgaacgactt agcgggcatg ttggcgcaga
     2761 attggcagcc ttgggacaac atgggcatcc cggcgtggct gggctctttt cagaaagagt
     2821 cgcgggaagt tggcctgctt gcgggtatga cctggcttcc gcgtaaagtt tacctgcacg
     2881 atccggttcc gggtcaggct gcgtgctgca gctgtggttt gccgagtgaa gctctggttt
     2941 actcatgctc cattgaggtg gaaccagtgc caaagggctt ggagtggaag gacccgcacg
     3001 gcgtttacac cgaccagggc aaatccctgc agtccaaaat caagctgatg agcaatgacc
     3061 gctatacctt cgcggaccgt gactggtatt ctccgttgtt ctcgtatctt cacgccgaag
     3121 gtaatagccg ccagggtaag ctgtggctgg tgggttttgc gagcgataaa gcaaaatcta
     3181 tcgacatctg ggataagatc atcgagctgg agggcaccga caccaatgac gaactgttgg
     3241 cgcaactcgc caatcgtgca accgcgctga atgcaatgcg taaaaagccg ctacgcggtg
     3301 attttaaaaa gagcgttggc accccgcaga ttgcggatat catcccgcat gcggaaaacc
     3361 gcatcgccat taacgcaggc aaaatgacgg aaaaccgtgg ttactcttgg caagatgccg
     3421 atacggagta cggagaactg ctgaccaagg tggcatactc gctggagccg gcgcaaacgg
     3481 tggatgctcg tctgaagcgc ggtaacttta tcagccgtaa gccgtggccg attatccccg
     3541 agagcaaaac aaaacccgca gagggggacc agaatgaata gggatcttga ggccgctcga
     3601 gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
     3661 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     3721 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     3781 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     3841 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     3901 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3961 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     4021 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     4081 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     4141 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     4201 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     4261 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     4321 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     4381 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     4441 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     4501 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     4561 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     4621 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     4681 aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg
     4741 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     4801 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     4861 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     4921 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     4981 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     5041 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     5101 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     5161 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     5221 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     5281 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     5341 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     5401 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     5461 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     5521 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     5581 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     5641 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     5701 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     5761 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     5821 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     5881 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     5941 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     6001 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     6061 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     6121 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     6181 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     6241 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     6301 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     6361 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     6421 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     6481 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     6541 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     6601 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6661 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6721 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6781 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6841 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6901 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6961 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     7021 tatatgagta aacttggtct gacagttacc aatgctt
//
