LOCUS       sequence-416239-        6214 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FLAG-SV40 NLS-Cas5 from JAGBLH010000220 Cas8-HNH in pcDNA3.1(+).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6214)
  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 6214)
  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: pHS1862
FEATURES             Location/Qualifiers
     source          1..6214
                     /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(2790..2807)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2796..3020
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3066..3494
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3153..3172)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3363..3384
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3503..3523)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3508..3837
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3688..3823
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3750..3769
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3904..4698
                     /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(3958..3977)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4568..4587
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4872..4993
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4909..4928)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4963..4982
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5042..5058)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5042..5058)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5055..5077)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5066..5082
                     /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(5090..5096,5097..5114,5115..5120))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5135..5156
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5273..5290)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5444..6032)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5524..5543)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6203..6214,1..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 tccatgaagg
     1981 gatatatact gcttgaaaaa gtaaatattg aaaacgctaa tgcatttaat aacattatag
     2041 taggaatccc ggctatcacc tcattcctgg gctttgcaag agcgctagag cgcaaactga
     2101 acgcgaaaga gatcgctatc cgtatcaacg gtgttggcct ggaatttcat gaatatgaat
     2161 taaaaggtta caagaacaaa cgtggtcagt atgtgaccag ctgtccgctg ccgggttcca
     2221 ttccaggtca gaacgagaag aagctggacg ctcacatcat gaatcaggca tatattgatc
     2281 tgaatatgag ctttttgctg gaggtggagg gtccgcatgt tgatatgtct acgtgcaaat
     2341 ctattaaatc gaccatggaa acgctgcgta ttgcgggtgg cattattcgc aactacaaga
     2401 agatccgctt gattgacacc cttgccgata tcccgtacgg ctacttcttg accctccgtc
     2461 aggataatct gaatgatgcg gcaggcgacg acatgctgga taagatgatt catgcgttgc
     2521 aacaagagga cactttggtt ccgattgccg ttggcttcaa ggcgctgagc gaagtcggtc
     2581 acgtggaggg ccaacgtgat ccggaaaagg accactgctt cgtggagtcc atcttcagcc
     2641 tgggcggttt tgaatgtagc aaaatcctgg aggacatcaa cagctgcctg tggcgctata
     2701 aaacagaaga agggctatat ctttgcacca taatttgagg atcctgaggc cgctcgagtc
     2761 tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     2821 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     2881 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     2941 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     3001 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     3061 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     3121 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     3181 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3241 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     3301 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     3361 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     3421 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     3481 atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     3541 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     3601 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     3661 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     3721 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc
     3781 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     3841 ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt
     3901 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     3961 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     4021 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     4081 ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     4141 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     4201 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     4261 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     4321 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     4381 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc
     4441 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     4501 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     4561 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     4621 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     4681 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     4741 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     4801 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     4861 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4921 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     4981 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     5041 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     5101 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     5161 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     5221 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     5281 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     5341 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     5401 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     5461 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     5521 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     5581 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     5641 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     5701 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     5761 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     5821 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5881 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     5941 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     6001 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     6061 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     6121 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     6181 atgagtaaac ttggtctgac agttaccaat gctt
//
