LOCUS       sequence-416242-        6457 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FLAG-SV40 NLS-Csy3/Cas7 from JAGBLH010000220 Cas8-HNH in 
            pcDNA3.1(+).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6457)
  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 6457)
  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: pHS1859
FEATURES             Location/Qualifiers
     source          1..6457
                     /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"
     CDS             2667..2681
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     primer_bind     complement(3033..3050)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3039..3263
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3309..3737
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3396..3415)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3606..3627
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3746..3766)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3751..4080
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3931..4066
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3993..4012
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4147..4941
                     /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(4201..4220)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4811..4830
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5115..5236
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5152..5171)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5206..5225
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5285..5301)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5285..5301)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5298..5320)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5309..5325
                     /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(5333..5339,5340..5357,5358..5363))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5378..5399
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5516..5533)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5687..6275)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5767..5786)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6446..6457,1..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 tccatggcag
     1981 ctaataaaaa ggcaaccaat gtcacgctaa aatcccgccc cgaaaatcta tcatttgcta
     2041 ggtgtttaaa cacgactgag gcgaaattct ggcagaccga ttttctgaaa cgccatacgt
     2101 tcaaattacc gctgctgatt accgataagg cggtcctcgc gagcaaaggc cacgaaatgc
     2161 cgcctgataa gctggaaaaa gagatcatgg acccgaatcc gcaaaagtcc cagtcttgta
     2221 ccctgtcgac cgagtgcgat acgctgcgta ttgactttgg tattaaagtg ctgccggtta
     2281 aagaaagcat gtacagctgc agcgattaca actatcgtac cgcaatttat cagaaaatcg
     2341 acgaatacat cgcggaggac ggcttcttga ccttggcgaa gcgctatgtg aataacatcg
     2401 cgaatgcccg ttttttgtgg cgtaatcgta aaggtgctga gatcatcgaa accattgtta
     2461 ccattgagga caaggagtac ccgagcttta atagcaagtc cttcaacctg gatactttcg
     2521 tggaggacaa cgccaccatc aacgaaatcg ctcagcagat tgccgatacg tttgctggca
     2581 agcgcgaata cttgaatatt tacgtcacct gctttgttaa gatcggttgt gcaatggaag
     2641 tgtatccatc gcaagaaatg acctttgatg acgatgacaa gggtaagaaa ctgttcaagt
     2701 tcgagggtag cgcaggtatg catagccaaa agatcaacaa cgcgctgcgt accattgaca
     2761 catggtatcc ggactacacc acctatgaat ttccgattcc ggtagagaac tacggcgcgg
     2821 cgcgttcaat cggcatcccg ttcagaccag acaccaaatc tttctataaa cttatcgacc
     2881 gcatgattct gaaaaacgag gatctgccga tcgaggacaa gcactacgtt atggcaattt
     2941 tgattcgagg cggcatgttc agcaagaaac aagaaaaata aggatcctga ggccgctcga
     3001 gtctaaaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
     3061 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     3121 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     3181 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     3241 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     3301 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3361 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     3421 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3481 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     3541 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3601 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     3661 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     3721 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3781 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     3841 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3901 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3961 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     4021 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     4081 aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg
     4141 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     4201 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     4261 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     4321 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     4381 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     4441 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     4501 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     4561 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     4621 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     4681 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     4741 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     4801 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     4861 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     4921 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     4981 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     5041 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     5101 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     5161 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     5221 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     5281 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     5341 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     5401 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5461 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5521 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5581 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5641 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5701 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5761 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5821 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5881 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5941 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     6001 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6061 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6121 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6181 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6241 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6301 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6361 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6421 tatatgagta aacttggtct gacagttacc aatgctt
//
