LOCUS       sequence-495662-        6302 bp    DNA     circular SYN 17-DEC-2025
DEFINITION  LAP2 fused to BFP and NLS, a nuclear localization signal. Imaging 
            shows primarily nucleolar localization..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6302)
  TITLE     Ting Lab LplA Plasmids 2025
REFERENCE   2  (bases 1 to 6302)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 17, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: His-LAP-BFP-NLS
FEATURES             Location/Qualifiers
     source          1..6302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             922..939
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(1006..1008,1009..1011,1012..1722)
                     /codon_start=1
                     /product="blue variant of GFP"
                     /label=BFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1051..1072)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1312..1331)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1659..1680
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1723..1743
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1747..1767
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1771..1791
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(1855..1873)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1856..1873)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1893..1910)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1899..2123
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2169..2597
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2256..2275)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2466..2487
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2606..2626)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2611..2940
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2791..2926
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2853..2872
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3007..3801
                     /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(3061..3080)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3671..3690
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3975..4096
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4012..4031)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4066..4085
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4145..4161)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4145..4161)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4158..4180)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4169..4185
                     /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(4193..4199,4200..4217,4218..4223))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4238..4259
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4376..4393)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4547..5135)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4627..4646)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5306..6097,6098..6166))
                     /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     5929..5948
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6167..6271)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc
      901 gccaccatgg tgcggggttc tcatcatcat catcatcatg gtatggctag cggcttcgag
      961 atcgacaagg tgtggtacga cctggacgcc aaggatccta gctcgatggt gagcaagggc
     1021 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     1081 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     1141 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     1201 agccacggcg tccagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     1261 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     1321 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     1381 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     1441 ttcaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     1501 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     1561 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca gccactacct gagcacccag
     1621 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     1681 accgccgccg ggatcactct cggcatggac gagctgtaca agccaaaaaa gaagagaaag
     1741 gtagatccaa aaaagaagag aaaggtagat ccaaaaaaga agagaaaggt ataagaattc
     1801 tgcagatatc catcacactg gcggccgctc gagcatgcat ctagagggcc ctattctata
     1861 gtgtcaccta aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     1921 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     1981 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     2041 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     2101 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     2161 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     2221 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     2281 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     2341 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     2401 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     2461 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     2521 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     2581 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     2641 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     2701 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     2761 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     2821 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     2881 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     2941 aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg
     3001 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     3061 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     3121 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     3181 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     3241 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     3301 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     3361 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     3421 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     3481 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     3541 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     3601 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     3661 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     3721 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     3781 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     3841 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     3901 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     3961 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     4021 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     4081 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     4141 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4201 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     4261 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4321 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4381 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4441 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4501 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4561 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4621 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4681 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4741 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4801 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4861 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4921 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4981 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5041 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5101 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5161 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5221 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5281 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5341 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5401 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5461 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5521 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5581 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     5641 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5701 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     5761 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     5821 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     5881 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     5941 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6001 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6061 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6121 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6181 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6241 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     6301 tc
//
