LOCUS       sequence-416244-        6616 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FLAG-SV40 NLS-Cas5-HNH from MWEE01000013 Cas5-HNH in pcDNA3.1(+).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6616)
  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 6616)
  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: pHS1517
FEATURES             Location/Qualifiers
     source          1..6616
                     /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(3192..3209)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3198..3422
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3468..3896
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3555..3574)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3765..3786
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3905..3925)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3910..4239
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4090..4225
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4152..4171
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4306..5100
                     /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(4360..4379)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4970..4989
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5274..5395
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5311..5330)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5365..5384
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5444..5460)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5444..5460)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5457..5479)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5468..5484
                     /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(5492..5498,5499..5516,5517..5522))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5537..5558
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5675..5692)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5846..6434)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5926..5945)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6605..6616,1..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 tctatgagtg
     1981 cgccacccaa caccttattc ttgaggttag aaggggctct acaatcatgg ggatcgaacg
     2041 aagcgaagtt cgccctgcgc cggaccgcag acgcgccaac taaatccggc gttctgggcc
     2101 tgctgtgcgc ggctatgggc atcggtcgtg ccgaggcggc ggactcttgg ctgccgaaac
     2161 tggctaatct gcgtatgggt gttcgtatcg atcgtccggg catccgctgg tgggattttc
     2221 acaccgtagg cgcaggccaa cgtatgcgta tggcggagct gaaggcgcca aagaagccga
     2281 gcatggtggg cgcagcgctg gcggaaaccc tgaccccgag caaagtcaaa acccgtgcgg
     2341 aaaccttgtt gtcccgccgt gagtacctcg cggacgctag cttcctggtg gcgttgcaag
     2401 gtgagccgga acttgtcgca aagctgtcgg cggccttagc caaaccggtt tgggcgattt
     2461 atttgggtcg taagagctgt ccgccatctc gccccgtgtg cgaacatccg ccgggctttt
     2521 ataacaccct ggaagaagcg ctgagcgcag ttccgctaca aaaacgttgg cataacgagc
     2581 cgctgccgca gattctgccg tgtgttatgg actggattcc gggttatgat ggtgagcacg
     2641 ctccggatga tgcggagatc cactacgact tgccggtgag cttccagccg cctcgtcatc
     2701 tgccgcgttt tgttattcgt cgggaactgg tcgtgggtga ggacgttcag gtttccagag
     2761 aaactggtac gagcgtctgg cgtcctaagg gtacgcgtgc ggactacaac aacagcgaat
     2821 acaaaaaggt gcgcgccgag cgcctggtga tggatcacgc tgcctgcatg gtttgcaaag
     2881 caccggcaac caccgtgcag cacgtgaatt atcgtcgcgc tggcggtaaa gagatcccgg
     2941 aggatttaag agctttgtgc cgcctgtgcc atgacgcgtg taccatgtta gaatacggct
     3001 ctggtatgac cacgaatcgt atcgacccgt gcgatccgat ttggagagaa cgtatcctcg
     3061 ctaagcgcaa ggagattgtt gaatttcgta gccgcggtca gcgtttccgc aaaatgaagc
     3121 cagaggagga aaacggatga ggatcttgag gccgctcgag tctagagggc ccgtttaaac
     3181 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     3241 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     3301 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     3361 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     3421 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     3481 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3541 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3601 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3661 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     3721 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3781 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     3841 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     3901 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     3961 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     4021 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     4081 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     4141 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     4201 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     4261 tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     4321 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     4381 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     4441 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     4501 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     4561 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     4621 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     4681 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     4741 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     4801 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     4861 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     4921 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     4981 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     5041 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     5101 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     5161 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     5221 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     5281 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     5341 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     5401 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     5461 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     5521 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     5581 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     5641 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     5701 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     5761 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5821 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5881 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5941 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     6001 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     6061 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     6121 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     6181 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     6241 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     6301 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     6361 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     6421 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     6481 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     6541 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     6601 acagttacca atgctt
//
