LOCUS       sequence-392744-        7943 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression for SV40 NLS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7943)
  AUTHORS   Price RM, Budzynski MA, Shen J, Mitchell JE, Kwan JZJ, Teves SS
  TITLE     Heat shock transcription factors demonstrate a distinct mode of 
            interaction with mitotic chromosomes.
  JOURNAL   Nucleic Acids Res. 2023 Apr 28:gkad304. doi: 10.1093/nar/gkad304.
  PUBMED    37114996
REFERENCE   2  (bases 1 to 7943)
  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: "Nucleic 
            Acids Res. 2023 Apr 28:gkad304. doi: 10.1093/nar/gkad304."
COMMENT     Sequence Label: NLS-Halo
FEATURES             Location/Qualifiers
     source          1..7943
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     481..500
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(719..823)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(849..1304)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(986..1007)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1198..1217
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1445..1462
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1446..1462
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   1592..1654
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        2108..2319
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2332..2600
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      2626..2635
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2635..2700
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2704..2724
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2728..2748
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2752..2772
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2779..3669
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNRVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             3682..3702
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     misc_feature    3733..4305
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3900..3917)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4127..4146
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4314..5108
                     /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
                     VLFVKTDLSGALNELQDEAARLSWLAATGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4368..4387)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4978..4997
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5168..5188)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    5230..5351
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5267..5286)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5321..5340
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   6115..6149
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6640..6656)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6640..6656)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6653..6675)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6664..6680
                     /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(6688..6694,6695..6712,6713..6718))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6733..6754
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6871..6888)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7042..7630)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7122..7141)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7801..7943,1..649,650..718))
                     /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 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
       61 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
      121 ctagagtaag tagttcgccg gttaatagtt tacgcaacgt tgttgccatt gctacaggca
      181 tcgtggtgtc gcgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
      241 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
      301 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
      361 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
      421 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
      481 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
      541 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
      601 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
      661 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
      721 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
      781 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
      841 tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
      901 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaaggata
      961 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     1021 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     1081 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     1141 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     1201 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     1261 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccattcag
     1321 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc
     1381 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg
     1441 acattgtaaa acgacggcca gtgagcgcgc ctcgttcatt cacgtttttg aacccgtgga
     1501 ggacgggcag actcgcggtg caaatgtgtt ttacagcgtg atggagcaga tgaagatgct
     1561 cgacacgctg cagaacacgc agctagatta accctagaaa gataatcata ttgtgacgta
     1621 cgttaaagat aatcatgtgt aaaattgacg catgtgtttt atcggtctgt atatcgaggt
     1681 ttatttatta atttgaatag atattaagtt ttattatatt tacacttaca tactaataat
     1741 aaattcaaca aacaatttat ttatgtttat ttatttatta aaaaaaacaa aaactcaaaa
     1801 tttcttctat aaagtaacaa aacttttatg agggacagcc ccccccccaa agcccccagg
     1861 gatgtaatta cgtccctccc ccgctagggg gcagcagcga gccgcccggg gctccgctcc
     1921 ggtccggcgc tccccccgca tccccgagcc ggcagcgtgc ggggacagcc cgggcacggg
     1981 gaaggtggca cgggatcgct ttcctctgaa cgcttctcgc tgctctttga gcctgcagac
     2041 acctgggggg atacggggaa aaggcctcca cggccaagga tctgcgatcg ctccggtgcc
     2101 cgccagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc
     2161 aattgaacgg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
     2221 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg
     2281 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagc tgaagcttcg aggggctcgc
     2341 atctctcctt cacgcgcccg ccgccctacc tgaggccgcc atccacgccg gttgagtcgc
     2401 gttctgccgc ctcccgcctg tggtgcctcc tgaactgcgt ccgccgtcta ggtaagttta
     2461 aagctcaggt cgagaccggg cctttgtccg gcgctccctt ggagcctacc tagactcagc
     2521 cggctctcca cgctttgcct gaccctgctt gctcaactct acgtctttgt ttcgttttct
     2581 gttctgcgcc gttacagatc caagctgtga ccggcgccta ctctagccac catggactac
     2641 aaagaccatg acggtgatta taaagatcat gacatcgatt acaaggatga cgatgacaag
     2701 cttccaaaaa agaagagaaa ggtagatccc aagaaaaaga gaaaggtcga tccaaagaag
     2761 aaaagaaaag tgggctccat ggcagaaatc ggtactggct ttccattcga cccccattat
     2821 gtggaagtcc tgggcgagcg catgcactac gtcgatgttg gtccgcgcga tggcacccct
     2881 gtgctgttcc tgcacggtaa cccgacctcc tcctacgtgt ggcgcaacat catcccgcat
     2941 gttgcaccga cccatcgctg cattgctcca gacctgatcg gtatgggcaa atccgacaaa
     3001 ccagacctgg gttatttctt cgacgaccac gtccgcttca tggatgcctt catcgaagcc
     3061 ctgggtctgg aagaggtcgt cctggtcatt cacgactggg gctccgctct gggtttccac
     3121 tgggccaagc gcaatccaga gcgcgtcaaa ggtattgcat ttatggagtt catccgccct
     3181 atcccgacct gggacgaatg gccagaattt gcccgcgaga ccttccaggc cttccgcacc
     3241 accgacgtcg gccgcaagct gatcatcgat cagaacgttt ttatcgaggg tacgctgccg
     3301 atgggtgtcg tccgcccgct gactgaagtc gagatggacc attaccgcga gccgttcctg
     3361 aatcgtgttg accgcgagcc actgtggcgc ttcccaaacg agctgccaat cgccggtgag
     3421 ccagcgaaca tcgtcgcgct ggtcgaagaa tacatggact ggctgcacca gtcccctgtc
     3481 ccgaagctgc tgttctgggg caccccaggc gttctgatcc caccggccga agccgctcgc
     3541 ctggccaaaa gcctgcctaa ctgcaaggct gtggacatcg gcccgggtct gaatctgctg
     3601 caagaagaca acccggacct gatcggcagc gagatcgcgc gctggctgtc gacgctcgag
     3661 atttccggcg agccaaccac tgaggatctg tactttcaga gcgataactc cggactcaga
     3721 tccgcggccg cgcccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga
     3781 ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa
     3841 tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc
     3901 tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc
     3961 ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg
     4021 cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca
     4081 accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag
     4141 cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct
     4201 ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc
     4261 cccgaaccac ggggacgtgg ttttcctttg aaaaacacga taatacctcc ggaatgattg
     4321 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     4381 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     4441 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     4501 aggcagcgcg gctatcgtgg ctggccgcga cgggcgttcc ttgcgcagct gtgctcgacg
     4561 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     4621 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     4681 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     4741 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     4801 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     4861 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     4921 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     4981 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     5041 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     5101 tcttctgagt cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     5161 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     5221 cgttaactaa acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     5281 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     5341 aatgtatctt atcatgtctg gaattgactc aaatgatgtc aattagtcta tcagaagctc
     5401 atctggtctc ccttccgggg gacaagacat ccctgtttaa tatttaaaca gcagtgttcc
     5461 caaactgggt tcttatatcc cttgctctgg tcaaccaggt tgcagggttt cctgtcccca
     5521 caggaacgaa gtccctaaag aaacagtggc agccaggttt agccccggaa ttgactggat
     5581 tcctttttta gggcccattg gtatggcttt ttccccgtat ccccccaggt gtctgcaggc
     5641 tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg tgccaccttc cccgtgcccg
     5701 ggctgtcccc gcacgctgcc ggcacgggga tgcgggggga gcgccggacc ggagcggagc
     5761 cccgggcggc tcgctgccgc cccctagcgg gggagggacg taattacatc cctgggggct
     5821 ttgggggggg ggctgtcccc gatatctata acaagaaaat atatatataa taagttatca
     5881 cgtaagtaga acatgaaata acaatataat tatcgtatga gttaaatctt aaaagtcacg
     5941 taaaagataa tcatgcgtca ttttgactca cgcggtcgtt atagttcaaa atcagtgaca
     6001 cttaccgcat tgacaagcac gcctcacggg agctccaagc ggcgactgag atgtcctaaa
     6061 tgcacagcga cggattcgcg ctatttagaa agagagagca atatttcaag aatgcatgcg
     6121 tcaattttac gcagactatc tttctagggt taatctagct gcatcaggat catatcgtcg
     6181 ggtctttttt ccggctcagt catcgcccaa gctggcgcta tctgggcatc ggggaggaag
     6241 aagcccgtgc cttttcccgc gaggttgaag cggcatggaa agagtttgcc gaggatgact
     6301 gctgctgcat tgacgttgag cgaaaacgca cgtttaccat gatgattcgg gaaggtgtgg
     6361 ccatgcacgc ctttaacggt gaactgttcg ttcaggccac ctgggatacc agttcgtcgc
     6421 ggcttttccg gacacagttc cggatggtca gcccgaagcg catcagcaac ccgaacaata
     6481 ccggcgacag ccggaactgc cgtgccggtg tgctgattaa tgacagcggt gcggcgctgg
     6541 gatattacgt cagcgaggac gggtatcctg gctggatgcc gcagaaatgg acatggttac
     6601 cccgtgagtt acccggcggg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     6661 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     6721 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     6781 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     6841 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     6901 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     6961 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     7021 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     7081 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     7141 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     7201 cgcctttctc ccttcgggga gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     7261 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     7321 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     7381 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     7441 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     7501 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     7561 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     7621 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     7681 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcgcct agatcctttt
     7741 aaattaaaaa tgaagtctta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     7801 ttaccaatgt ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     7861 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     7921 cagtgctgca atgataccgc gag
//
