LOCUS       sequence-345519-        6868 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of nuclear localized AwaIscB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6868)
  AUTHORS   Altae-Tran H, Kannan S, Demircioglu FE, Oshiro R, Nety SP, McKay LJ,
            Dlakic M, Inskeep WP, Makarova KS, Macrae RK, Koonin EV, Zhang F
  TITLE     The widespread IS200/IS605 transposon family encodes diverse 
            programmable RNA-guided endonucleases.
  JOURNAL   Science. 2021 Oct;374(6563):57-65. doi: 10.1126/science.abj6856. 
            Epub 2021 Sep 9.
  PUBMED    34591643
REFERENCE   2  (bases 1 to 6868)
  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"; doi: 
            "10.1126/science.abj6856"; journalName: "Science"; date: "2021-10"; 
            volume: "374"; issue: "6563"; pages: "57-65"
COMMENT     Sequence Label: pHS0781 pcDNA3.1(+) CMV-SV40
            NLS-AwaIscB-nucle...
FEATURES             Location/Qualifiers
     source          1..6868
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      683..692
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             698..718
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2045..2092
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             2099..2179
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(2225..2242)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2231..2455
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2501..2929
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2588..2607)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2798..2819
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2938..2958)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2943..3272
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3123..3258
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3185..3204
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3339..4133
                     /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(3393..3412)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4003..4022
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4307..4428
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4344..4363)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4398..4417
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4477..4493)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4477..4493)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4490..4512)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4501..4517
                     /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(4525..4531,4532..4549,4550..4555))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4570..4591
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4708..4725)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4879..5467)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4959..4978)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5638..6429,6430..6498))
                     /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"
     primer_bind     6261..6280
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6499..6603)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6678..6697)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcgttt aaacttaagc ttgccaccat gggatctcca aaaaagaaac ggaaggtggg
      721 atctagcgtg ttcgtgctga acaagcagaa acggcctctg atgccctgca gcgagaagag
      781 agcccgcctg ctgctggaaa gaggcagagc cgtggtggtg cggctggtgc cattcaccat
      841 ccggctgaag gatagaatcg gcggcgtgct gcagcccctg agactgaagc tggaccctgg
      901 ctctaagaca accggcatcg ccctggtgcg cgaggtggtg cggcgggacg agagcgtggt
      961 ttggctggct gaactgacac acagaggata tcagatcagc gaggccctga gagctagaag
     1021 cgccatgaga agaagaagaa ggtccgccaa cctgcggtac agagccccta gattcctgaa
     1081 tcggaccaag cctaagggct ggctggcccc tagcctgaga catagagtgg aaaccaccat
     1141 taactgggtc aagagactgc ggagactggc tcctatcacg gaaatcaccc aggagctggt
     1201 caggttcgac ctgcaagcca tgcagcaccc cgagatcagc ggcattgagt accagcaggg
     1261 cgagctggct ggctacgagg tgcgggaata cctgctggaa aagtggcagc ggacatgcgc
     1321 ctactgcggc gcccagcagg tgcccctcca gatcgagcac atcagaccta agtccgccgg
     1381 cggaagcaac agattgtcta atctcaccct ggcttgcgcc ccttgcaacc acaagaaggg
     1441 cgctcaatct atcgaggcct tcctgaagca caaactggaa ctgctgaaac agatccaggc
     1501 ccaggcacaa gcccctctga aagacgccgc cgccgtgaac accacccggt gggccctgtt
     1561 caacgccctg aaggctacag gcctgcaggt gaaaaccggc agcggcggac agaccaaata
     1621 caaccgccag agactgggca tcccaaagac ccacgccctg gacgccgctt gtgtgggcaa
     1681 gctggatgcc ctgcacaact ggcagatccc tacactggcc atcaaggcga tgggcagagg
     1741 cagctaccag aggaccagac tgaatagatt cggcttcccc agaggccacc tgatgagaca
     1801 caagagaatc cacggttttc agaccggcga cagagtgatc gcacacatcc ccagcggcaa
     1861 aaaggccgga gtgcacgtgg gaagagtggc cgtccgaacc agcggatctt ttaacatcca
     1921 aacagccaca ggcgtgatcc agggcatcgc ccaccggcat tgtagcgtcc tgcagcgggc
     1981 cgacggctac ggctacagct tcaacctgac ccagcctgag gaagccagac tggctgccgg
     2041 atccaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa agggatctta
     2101 cccatacgat gttccagatt acgcttaccc atacgatgtt ccagattacg cttacccata
     2161 cgatgttcca gattacgctt gaggccgctc gagtctagag ggcccgttta aacccgctga
     2221 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     2281 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     2341 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     2401 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     2461 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     2521 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2581 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2641 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2701 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     2761 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     2821 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     2881 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
     2941 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     3001 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     3061 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     3121 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     3181 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
     3241 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
     3301 tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     3361 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     3421 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     3481 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     3541 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     3601 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     3661 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     3721 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     3781 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     3841 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     3901 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     3961 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     4021 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     4081 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     4141 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     4201 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     4261 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     4321 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     4381 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     4441 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     4501 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     4561 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     4621 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4681 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4741 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4801 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4861 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4921 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4981 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5041 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     5101 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5161 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5221 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5281 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     5341 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5401 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5461 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5521 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5581 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5641 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     5701 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     5761 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     5821 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     5881 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     5941 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6001 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6061 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     6121 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6181 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6241 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     6301 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6361 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6421 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6481 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6541 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6601 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc
     6661 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
     6721 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
     6781 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
     6841 cgcgatgtac gggccagata tacgcgtt
//
