LOCUS       sequence-356068-        5779 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes NLS-2xBFP(4C), which is a tandem BFP with an N-terminal SV40
            NLS. The protein has 4 solvent accessible cysteines for labeling 
            with maleimide dyes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5779)
  AUTHORS   Chowdhury R, Sau A, Musser SM
  TITLE     Super-resolved 3D tracking of cargo transport through nuclear pore 
            complexes.
  JOURNAL   Nat Cell Biol. 2022 Jan;24(1):112-122. doi: 
            10.1038/s41556-021-00815-6. Epub 2022 Jan 10.
  PUBMED    35013558
REFERENCE   2  (bases 1 to 5779)
  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: 
            "10.1038/s41556-021-00815-6"; journalName: "Nat Cell Biol"; date: 
            "2022-01"; volume: "24"; issue: "1"; pages: "112-122"
COMMENT     Sequence Label: pNLS-2xBFP(4C)
FEATURES             Location/Qualifiers
     source          1..5779
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        16..93
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             94..1176
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(113..132)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1408..1413,1414..1430,1431..1437)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1445..1461
                     /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)."
     primer_bind     1450..1472
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    1478..1547
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             1598..1624
                     /codon_start=1
                     /product="synthetic cistron containing a ribosome binding 
                     site (Shine-Dalgarno sequence), for enhancing the bacterial
                     expression of a downstream cistron (Schoner, 1997)"
                     /label=minicistron
                     /note="This first cistron should terminate 3 bp upstream of
                     the ATG for the second cistron."
                     /translation="MYRLNKEE"
     CDS             1643..1663
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1673..2383
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFTHGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKAN
                     FKIRHNIEDGCVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             2393..3103
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFTHGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKAN
                     FKIRHNIEDGCVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             3119..3136
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3178..3195)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3348..3434
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3526..3553
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3572..3663
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3664..3732,3733..4524)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3882..3901)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4695..5283
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5184..5203
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5437..5454
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5469..5609
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5535..5554)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5695..5717
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gcatgcattt acgttgacac catcgaatgg tgcaaaacct ttcgcggtat ggcatgatag
       61 cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc
      121 gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac
      181 gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc
      241 aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc
      301 agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa
      361 ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg
      421 gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat
      481 gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat
      541 gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg
      601 ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta
      661 agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa
      721 attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc
      781 atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg
      841 gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg
      901 gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc
      961 aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag
     1021 ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc
     1081 ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     1141 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta
     1201 gcgcgaattg atctggtttg acagcttatc atcgactgca cggtgcacca atgcttctgg
     1261 cgtcaggcag ccatcggaag ctgtggtatg gctgtgcagg tcgtaaatca ctgcataatt
     1321 cgtgtcgctc aaggcgcact cccgttctgg ataatgtttt ttgcgccgac atcataacgg
     1381 ttctggcaaa tattctgaaa tgagctgttg acaattaatc atccggctcg tataatgtgt
     1441 ggaattgtga gcggataaca atttcacaca ggaaacagcg ccgctgagaa aaagcgaagc
     1501 ggcactgctc tttaacaatt tatcagacaa tctgtgtggg cactcgaccg gaattatcga
     1561 ttaactttat tattaaaaat taaagaggta tatattaatg tatcgattaa ataaggagga
     1621 ataaaccatg agactcgagc ctccaaagaa gaaaagaaag gtcgacagcg cgagcaaagg
     1681 agaagaactc ttcactggag ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg
     1741 gcacaaattt tctgtcagtg gagagggtga aggtgatgca acatacggaa aacttaccct
     1801 taaatttatt tgcactactg gaaaactacc tgttccatgg ccaacacttg tcactacttt
     1861 cactcatggt gttcagtgct tttcaagata cccggatcat atgaaacggc atgacttttt
     1921 caagagtgcc atgcccgaag gttatgtaca ggaaagaact atatttttca aagatgacgg
     1981 gaactacaag acacgtgctg aagtcaagtt tgaaggtgat acccttgtta atagaatcga
     2041 gttaaaaggt attgatttta aagaagatgg aaacattctt gggcacaaat tggaatacaa
     2101 ctataactca cacaatgtat acatcatggc agacaaacaa aagaatggaa tcaaagctaa
     2161 cttcaaaatt agacacaaca ttgaagatgg atgcgttcaa ctagcagacc attatcaaca
     2221 aaatactcca attggcgatg gccctgtcct tttaccagac aaccattacc tgtccacaca
     2281 atctgccctt tcgaaagatc ccaacgaaaa gagagaccac atggtccttc ttgagtttgt
     2341 aacagctgct gggattacac atggcatgga tgaactatac aaaggatgcg ctagcaaagg
     2401 agaagaactc ttcactggag ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg
     2461 gcacaaattt tctgtcagtg gagagggtga aggtgatgca acatacggaa aacttaccct
     2521 taaatttatt tgcactactg gaaaactacc tgttccatgg ccaacacttg tcactacttt
     2581 cactcatggt gttcagtgct tttcaagata cccggatcat atgaaacggc atgacttttt
     2641 caagagtgcc atgcccgaag gttatgtaca ggaaagaact atatttttca aagatgacgg
     2701 gaactacaag acacgtgctg aagtcaagtt tgaaggtgat acccttgtta atagaatcga
     2761 gttaaaaggt attgatttta aagaagatgg aaacattctt gggcacaaat tggaatacaa
     2821 ctataactca cacaatgtat acatcatggc agacaaacaa aagaatggaa tcaaagctaa
     2881 cttcaaaatt agacacaaca ttgaagatgg atgcgttcaa ctagcagacc attatcaaca
     2941 aaatactcca attggcgatg gccctgtcct tttaccagac aaccattacc tgtccacaca
     3001 atctgccctt tcgaaagatc ccaacgaaaa gagagaccac atggtccttc ttgagtttgt
     3061 aacagctgct gggattacac atggcatgga tgaactatac aaaggatcct gcgagctcca
     3121 tcatcatcat catcattgaa agcttggctg ttttggcgga tgagagaaga ttttcagcct
     3181 gatacagatt aaatcagaac gcagaagcgg tctgataaaa cagaatttgc ctggcggcag
     3241 tagcgcggtg gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga
     3301 tggtagtgtg gggtctcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa
     3361 aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc
     3421 tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt
     3481 ggcgggcagg acgcccgcca taaactgcca ggcatcaaat taagcagaag gccatcctga
     3541 cggatggcct ttttgcgttt ctacaaactc ttttgtttat ttttctaaat acattcaaat
     3601 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     3661 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     3721 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     3781 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     3841 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     3901 tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     3961 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     4021 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     4081 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     4141 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     4201 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     4261 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     4321 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     4381 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     4441 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     4501 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     4561 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     4621 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     4681 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     4741 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     4801 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     4861 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     4921 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     4981 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     5041 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     5101 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     5161 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     5221 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     5281 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     5341 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     5401 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     5461 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     5521 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     5581 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     5641 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     5701 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag cagatcaatt
     5761 cgcgcgcgaa ggcgaagcg
//
