LOCUS       sequence-445256-        8528 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Mammalian expression of iPASnFR-L fused to Rab5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8528)
  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 8528)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-iPASnFR-L-mScarlet3-3xFLAG-Rab5
FEATURES             Location/Qualifiers
     source          1..8528
                     /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(4119..4136)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4125..4349
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4395..4823
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4482..4501)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4692..4713
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4832..4852)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4837..5166
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5017..5152
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5079..5098
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5233..6027
                     /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(5287..5306)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5897..5916
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6201..6322
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6238..6257)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6292..6311
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6371..6387)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6371..6387)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6384..6406)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6395..6411
                     /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(6419..6425,6426..6443,6444..6449))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6464..6485
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6602..6619)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6773..7361)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6853..6872)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7532..8323,8324..8392))
                     /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     8155..8174
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8393..8497)
                     /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 tcgtgcggta 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 gtttgttggc 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 gctcaagctt cgaattccga tggctagtcg aggcgcaaca agacccaacg ggccaaatac
     3481 tggaaataaa atatgccagt tcaaactagt acttctggga gagtccgctg ttggcaaatc
     3541 aagcctagtg cttcgttttg tgaaaggcca atttcatgaa tttcaagaga gtaccattgg
     3601 ggctgctttt ctaacccaaa ctgtatgtct tgatgacact acagtaaagt ttgaaatatg
     3661 ggatacagct ggtcaagaac gataccatag cctagcacca atgtactaca gaggagcaca
     3721 agcagccata gttgtatatg atatcacaaa tgaggagtcc tttgcaagag caaaaaattg
     3781 ggttaaagaa cttcagaggc aagcaagtcc taacattgta atagctttat cgggaaacaa
     3841 ggccgaccta gcaaataaaa gagcagtaga tttccaggaa gcacagtcct atgcagatga
     3901 caatagttta ttattcatgg agacatccgc taaaacatca atgaatgtaa atgaaatatt
     3961 catggcaata gctaaaaaat tgccaaagaa tgaaccacaa aatccaggag caaattctgc
     4021 cagaggaaga ggagtagacc ttaccgaacc cacacaacca accaggaatc agtgttgtag
     4081 taactaatct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     4141 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     4201 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     4261 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     4321 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     4381 tagggggtat ccccacgcgc cctgtagagg cgcattaagc gcggcgggtg tggtggttac
     4441 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     4501 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     4561 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     4621 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     4681 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     4741 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     4801 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     4861 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     4921 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     4981 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     5041 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     5101 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     5161 tgcaaaaagc tcccgggagc ttatatatcc attttcggat ctgatcaaga gacaggatga
     5221 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     5281 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     5341 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     5401 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     5461 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     5521 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     5581 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     5641 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     5701 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     5761 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     5821 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     5881 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     5941 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     6001 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     6061 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     6121 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     6181 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     6241 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     6301 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     6361 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     6421 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     6481 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     6541 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     6601 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     6661 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     6721 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     6781 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     6841 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     6901 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     6961 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     7021 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     7081 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     7141 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     7201 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     7261 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     7321 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     7381 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     7441 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     7501 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     7561 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     7621 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     7681 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     7741 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     7801 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     7861 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     7921 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     7981 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     8041 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     8101 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     8161 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     8221 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     8281 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     8341 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     8401 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     8461 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     8521 ctgacgtc
//
