LOCUS       sequence-445253-        7865 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of iPASnFR-H in the cytosol.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7865)
  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 7865)
  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-Cyto-iPASnFR-H-mScarlet3-3xFLAG
FEATURES             Location/Qualifiers
     source          1..7865
                     /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"
     primer_bind     1993..2014
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2117..2138)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             2672..3355
                     /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     3067..3086
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3356..3421
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3456..3473)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3462..3686
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3732..4160
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3819..3838)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4029..4050
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4169..4189)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4174..4503
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4354..4489
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4416..4435
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4570..5364
                     /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(4624..4643)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5234..5253
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5538..5659
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5575..5594)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5629..5648
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5708..5724)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5708..5724)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5721..5743)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5732..5748
                     /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(5756..5762,5763..5780,5781..5786))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5801..5822
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5939..5956)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6110..6698)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6190..6209)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6869..7660,7661..7729))
                     /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     7492..7511
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7730..7834)
                     /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 gctggctagc
      901 gtttaaactt aagcttgcca ccatgaacac gttgacggtt tacaattggg gtgattacat
      961 cgaccctgca ttaatccgta agttcgagaa gcaaacgggt ataaaggtta tatatcagac
     1021 gtttgactcc aatgaagcaa tgatggcaaa gattgctcaa ggtggagcaa cgttcgacgt
     1081 agcagttcca tctgagtacg cgatctctaa gatgaaggaa gatggattgc tgatcccctt
     1141 agaccattca aaattaccta acctcaaata cattaatccg cgcttcttaa atttaagctt
     1201 cgaccccgga aacagatact ctgtccccta cttctggggt accgtgggga tagtttacaa
     1261 tcccggcatg ttgggtggta agaagatcac ttcttggaac gacttgtggg atccagacct
     1321 gcgcaatcag atcctgcttg ttgacggagc tcgtgaggta atcggaatgg gacttaactc
     1381 actccactat tcactcaacg acaccaataa agagcatctc aagaaggcca aagagaagtt
     1441 ggacagactc gtgccaaatg tgaaagccat agtcggggat gaaatcaaga tgttgctggc
     1501 gaatgaggaa gcagcgatag gagttgtgtg gagtggggac gctgcggaga tcatcgccga
     1561 aaatgacaag ttggattacg tggtgcccaa agagggatcg aacttgtggt tcgacaatat
     1621 ggtgattcca aagaccgcga agaatattga aggagcacat aaattcatta acttcatgct
     1681 ggacccaaag aatgcggcgc agaacgccga gtacgttggc tattcaaccc caaataagaa
     1741 ggctttggat tacatgccta aggaaatcac agagtctaag aggcacacgg tctatatcac
     1801 cgccgacaag cagaagaacg gcatcaaggc gaacttcaag atccacctta acgtggagga
     1861 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     1921 gctgctgccc gacaaccact acctgagcta tcagtccgtg ctgagcaaag accccaacga
     1981 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc accgggaact ctctcggcat
     2041 ggacgagctg tacaagggcg gtaccggagg gagcatgagc aagggcgagg agctgttcac
     2101 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     2161 gcgcggcgag ggcgagggcg atgccaccaa cggcaagcta accctgaagt tcatctgcac
     2221 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgagct acggcgtgca
     2281 gtgcttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     2341 cgaaggctac gtccaggagc gcaccatcag cttcaaggac gacggaacct acaagacccg
     2401 cgccgaggtg aagttagagg gcgacaccct ggtgaaccgc atcgagctaa agggcatcga
     2461 cttcaaggag gacggcaaca tcctggggca caagctggag tacaacttta actatcatga
     2521 caagcggttc tatcccgatt tcgactcaat cggcaattta gaagtttaca agaatcttgg
     2581 aaagcgtatg ttggcttact acaatgaact gttcctgcaa tttaagatgc accgcaatgc
     2641 tgtgggccag gacacgcagg agcgtgaatt cgatagcacc gaggcagtga tcaaggagtt
     2701 catgcggttc aaggtgcaca tggagggctc catgaacggc cacgagttcg agatcgaggg
     2761 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaggg tgaccaaggg
     2821 tggccccctg cccttctcct gggacatcct gtcccctcag ttcatgtacg gctccagggc
     2881 cttcacgaag caccccgccg acatccccga ctactggaag cagtccttcc ccgagggctt
     2941 caagtgggag cgcgtgatga acttcgagga cggcggcgcc gtgtccgtgg cccaggacac
     3001 ctccctggag gacggcaccc tgatctacaa ggtgaagctc cgcggcacca acttccctcc
     3061 tgacggcccc gtaatgcaga agaagacaat gggctgggaa gcatccaccg agcggttgta
     3121 ccccgaggac gtcgtgctga agggcgacat taagatggcc ctgcgcctga aggacggcgg
     3181 ccgctacctg gcggacttca agaccaccta cagggccaag aagcccgtgc agatgcccgg
     3241 cgccttcaac atcgaccgca agttggacat cacatcccac aacgaggact acaccgtggt
     3301 ggaacagtac gaacgctccg tggcccgcca ctccaccggc ggctccggtg gctccgacta
     3361 caaagaccat gacggtgatt ataaagatca tgacatcgat tacaaggatg acgatgacaa
     3421 gtaatctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
     3481 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     3541 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     3601 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     3661 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     3721 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     3781 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     3841 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     3901 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     3961 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     4021 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     4081 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     4141 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     4201 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     4261 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     4321 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4381 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4441 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     4501 aaaaagctcc cgggagctta tatatccatt ttcggatctg atcaagagac aggatgagga
     4561 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     4621 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     4681 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     4741 aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     4801 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     4861 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     4921 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     4981 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     5041 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc
     5101 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     5161 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     5221 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     5281 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     5341 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     5401 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     5461 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     5521 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     5581 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     5641 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     5701 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     5761 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     5821 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5881 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     5941 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     6001 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     6061 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     6121 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     6181 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     6241 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     6301 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     6361 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     6421 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     6481 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     6541 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     6601 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     6661 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6721 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     6781 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     6841 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     6901 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     6961 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     7021 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     7081 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     7141 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     7201 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     7261 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     7321 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     7381 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     7441 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     7501 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     7561 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     7621 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     7681 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     7741 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     7801 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     7861 acgtc
//
