LOCUS       sequence-445258-        7998 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of iPASnFR-L in the nucleus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7998)
  AUTHORS   Tamura R, Chen J, De Jaeger M, Morris JF, Scott DA, Vangheluwe P, 
            Looger LL
  TITLE     Genetically encoded fluorescent sensors for visualizing polyamine 
            levels, uptake, and distribution
  JOURNAL   bioRxiv 2024.08.21.609037
REFERENCE   2  (bases 1 to 7998)
  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: "bioRxiv 
            2024.08.21.609037"
COMMENT     Sequence Label: pcDNA3.1-NLS-iPASnFR-L-mScarlet3-3xFLAG
FEATURES             Location/Qualifiers
     source          1..7998
                     /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      938..947
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             956..976
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             980..1000
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1004..1024
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     2104..2125
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2228..2249)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             2783..3466
                     /codon_start=1
                     /product="bright cysteine-free monomeric red fluorescent 
                     protein with fast and complete maturation (Gadella et al., 
                     2023)"
                     /label=mScarlet3
                     /translation="DSTEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKL
                     RVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYWKQSFPEGFKWERVMNFEDGGAV
                     SVAQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDVVLKGDIKMA
                     LRLKDGGRYLADFKTTYRAKKPVQMPGAFNIDRKLDITSHNEDYTVVEQYERSVARHST
                     GGSGGS"
     primer_bind     3178..3197
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3467..3532
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     promoter        complement(3551..3569)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3552..3569)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3589..3606)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3595..3819
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3865..4293
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3952..3971)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4162..4183
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4302..4322)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4307..4636
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4487..4622
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4549..4568
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4703..5497
                     /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(4757..4776)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5367..5386
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5671..5792
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5708..5727)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5762..5781
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5841..5857)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5841..5857)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5854..5876)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5865..5881
                     /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(5889..5895,5896..5913,5914..5919))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5934..5955
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6072..6089)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6243..6831)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6323..6342)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7002..7793,7794..7862))
                     /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     7625..7644
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7863..7967)
                     /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 gcttggtacc
      901 gagctcggat ccactagtaa cggccgccag tgtgctggcc accatgggat cagatccaaa
      961 aaagaagaga aaggtagacc cgaagaaaaa gcgtaaggtc gatcccaaaa agaaacggaa
     1021 agtgggatca ccggtcaaca cgttgacggt ttacaattgg ggtgattaca tcgaccctgc
     1081 attaatccgt aagttcgaga agcaaacggg tataaaggtt atatatcaga cgtttgactc
     1141 caatgaagca atgatggcaa agattgctca aggtggagca acgttcgacg tagcagttcc
     1201 atctgagtac gcgatctcta agatgaagga agatggattg ctgatcccct tagaccattc
     1261 aaaattacct aacctcaaat acattaatcc gcgcttctta aatttaagct tcgaccccgg
     1321 aaacagatac tctgtcccct acttctgggg taccgtgggg atagtttaca atcccggcat
     1381 gttgggtggt aagaagatca cttcttggaa cgacttgtgg gatccagacc tgcgcaatca
     1441 gatcctgctt gttgacggag ctcgtgcggt aatcggaatg ggacttaact cactccacta
     1501 ttcactcaac gacaccaata aagagcatct caagaaggcc aaagagaagt tggacagact
     1561 cgtgccaaat gtgaaagcca tagtcgggga tgaaatcaag atgttgctgg cgaatgagga
     1621 agcagcgata ggagttgtgt ggagtgggga cgctgcggag atcatcgccg aaaatgacaa
     1681 gttggattac gtggtgccca aagagggatc gaacttgtgg ttcgacaata tggtgattcc
     1741 aaagaccgcg aagaatattg aaggagcaca taaattcatt aacttcatgc tggacccaaa
     1801 gaatgcggcg cagaacgccg agtttgttgg ctattcaacc ccaaataaga aggctttgga
     1861 ttacatgcct aaggaaatca cagagtctaa gaggcacacg gtctatatca ccgccgacaa
     1921 gcagaagaac ggcatcaagg cgaacttcaa gatccacctt aacgtggagg acggcagcgt
     1981 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     2041 cgacaaccac tacctgagct atcagtccgt gctgagcaaa gaccccaacg agaagcgcga
     2101 tcacatggtc ctgctggagt tcgtgaccgc caccgggaac tctctcggca tggacgagct
     2161 gtacaagggc ggtaccggag ggagcatgag caagggcgag gagctgttca ccggggtggt
     2221 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgcgcggcga
     2281 gggcgagggc gatgccacca acggcaagct aaccctgaag ttcatctgca ccaccggcaa
     2341 gctgcccgtg ccctggccca ccctcgtgac caccctgagc tacggcgtgc agtgcttcgc
     2401 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     2461 cgtccaggag cgcaccatca gcttcaagga cgacggaacc tacaagaccc gcgccgaggt
     2521 gaagttagag ggcgacaccc tggtgaaccg catcgagcta aagggcatcg acttcaagga
     2581 ggacggcaac atcctggggc acaagctgga gtacaacttt aactatcatg acaagcggtt
     2641 ctatcccgat ttcgactcaa tcggcaattt agaagtttac aagaatcttg gaaagcgtat
     2701 gttggcttac tacaatgaac tgttcctgca atttaagatg caccgcaatg ctgtgggcca
     2761 ggacacgcag gagcgtgaat tcgatagcac cgaggcagtg atcaaggagt tcatgcggtt
     2821 caaggtgcac atggagggct ccatgaacgg ccacgagttc gagatcgagg gcgagggcga
     2881 gggccgcccc tacgagggca cccagaccgc caagctgagg gtgaccaagg gtggccccct
     2941 gcccttctcc tgggacatcc tgtcccctca gttcatgtac ggctccaggg ccttcacgaa
     3001 gcaccccgcc gacatccccg actactggaa gcagtccttc cccgagggct tcaagtggga
     3061 gcgcgtgatg aacttcgagg acggcggcgc cgtgtccgtg gcccaggaca cctccctgga
     3121 ggacggcacc ctgatctaca aggtgaagct ccgcggcacc aacttccctc ctgacggccc
     3181 cgtaatgcag aagaagacaa tgggctggga agcatccacc gagcggttgt accccgagga
     3241 cgtcgtgctg aagggcgaca ttaagatggc cctgcgcctg aaggacggcg gccgctacct
     3301 ggcggacttc aagaccacct acagggccaa gaagcccgtg cagatgcccg gcgccttcaa
     3361 catcgaccgc aagttggaca tcacatccca caacgaggac tacaccgtgg tggaacagta
     3421 cgaacgctcc gtggcccgcc actccaccgg cggctccggt ggctccgact acaaagacca
     3481 tgacggtgat tataaagatc atgacatcga ttacaaggat gacgatgaca agtaatctag
     3541 agggccctat tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc
     3601 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     3661 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     3721 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     3781 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     3841 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     3901 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     3961 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     4021 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     4081 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     4141 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     4201 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     4261 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg
     4321 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     4381 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     4441 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     4501 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     4561 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     4621 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga
     4681 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     4741 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     4801 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     4861 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
     4921 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     4981 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     5041 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     5101 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     5161 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     5221 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     5281 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     5341 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     5401 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     5461 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     5521 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     5581 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     5641 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     5701 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     5761 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     5821 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     5881 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     5941 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     6001 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     6061 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     6121 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     6181 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     6241 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     6301 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     6361 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     6421 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     6481 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     6541 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     6601 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta aagagttctt gaagtggtgg
     6661 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     6721 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     6781 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     6841 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     6901 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     6961 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     7021 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     7081 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     7141 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     7201 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     7261 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     7321 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     7381 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     7441 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     7501 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     7561 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     7621 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     7681 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     7741 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     7801 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     7861 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     7921 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     7981 aaagtgccac ctgacgtc
//
