LOCUS       sequence-381450-        6600 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of mutated human norpinephrine transporter tagged with 
            monomeric GFP at the N-terminus in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6600)
  AUTHORS   Luethi D, Maier J, Rudin D, Szollosi D, Angenoorth TJF, Stankovic S,
            Schittmayer M, Burger I, Yang JW, Jaentsch K, Holy M, Das AK, 
            Brameshuber M, Camacho-Hernandez GA, Casiraghi A, Newman AH, 
            Kudlacek O, Birner-Gruenberger R, Stockner T, Schutz GJ, Sitte HH
  TITLE     Phosphatidylinositol 4,5-bisphosphate (PIP2) facilitates 
            norepinephrine transporter dimerization and modulates substrate 
            efflux.
  JOURNAL   Commun Biol. 2022 Nov 17;5(1):1259. doi: 10.1038/s42003-022-04210-1.
  PUBMED    36396757
REFERENCE   2  (bases 1 to 6600)
  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: "Commun 
            Biol."; date: "2022-11-17"; volume: "5(1):1259. doi"; pages: " 
            10.1038/s42003-022-04210-1"
COMMENT     Sequence Label: mGFP-hNET-R121A/K334A/R440A
FEATURES             Location/Qualifiers
     source          1..6600
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAVGITLGMDELYK"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3388..3509
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3425..3444)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3479..3498
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3516..3971)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3653..3674)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3865..3884
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3998..4102
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4104..4461
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4104..4295
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4127..4147)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4312..4447
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4496..5290
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4496..5290
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4550..4569)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5160..5179
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5478..5497)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5522..5569
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5898..6486
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6387..6406
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc aaactgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgtcggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggactcag atctcgagct caagcttcga attccatgct tctggcgcgg
     1381 atgaacccgc aggtgcagcc cgagaacaac ggggcggaca cgggtccaga gcagcccctt
     1441 cgggcgcgca aaactgcgga gctgctggtg gtgaaggagc gcaacggcgt ccagtgcctg
     1501 ctggcgcccc gcgacggcga cgcgcagccc cgggagacct ggggcaagaa gatcgacttc
     1561 ctgctgtccg tagtcggctt cgcagtggac ctggccaacg tgtggcgctt cccctacctc
     1621 tgctacaaga acggcggcgg tgccttcttg atcccgtaca cactgttcct tatcatcgcg
     1681 gggatgcccc tgttctacat ggagctggct ctgggacagt acaacgcgga gggggctgcc
     1741 accgtttgga agatctgccc attcttcaaa ggcgttggct atgctgtcat cctgatcgcc
     1801 ctgtacgttg gcttctacta caacgtcatc atcgcctggt cactctacta cctcttctcc
     1861 tccttcaccc tcaacctgcc ctggaccgac tgtggccaca cctggaacag ccccaactgt
     1921 accgacccca agctcctcaa tggctccgtg cttggcaacc acaccaagta ctccaagtac
     1981 aagttcacgc cggcagccga gttttatgag cgtggtgtcc tgcaccttca cgagagcagc
     2041 gggattcatg acatcggcct gccccagtgg cagctcttgc tctgtctgat ggtcgtcgtc
     2101 atcgtcttgt attttagcct ctggaaaggg gtgaagacat caggaaaggt ggtgtggatc
     2161 acagccacgc tgccttactt cgtgctgttc gtgctcctgg tccatggggt caccctgccc
     2221 ggagcctcca atggcatcaa tgcctacctg cacatcgact tctaccgctt gaaagaggcc
     2281 acggtatgga ttgatgccgc aactcagata tttttttcct tgggggctgg atttggagta
     2341 ttgattgcat ttgccagtta caacgcattt gacaacaact gttacaggga tgccctgctg
     2401 accagcagca tcaactgtat caccagcttc gtctctgggt tcgccatctt ctccatcctt
     2461 ggttacatgg cccatgaaca caaggtcaac attgaggatg tggccacaga aggagctggc
     2521 ctagtgttca tcctgtatcc agaggccatt tctaccctgt ctggatctac attctgggct
     2581 gttgtgtttt tcgtcatgct cctggcgctg ggccttgaca gctcaatggg aggcatggag
     2641 gctgtcatca cgggcctggc cgatgacttc caggtcctga aggcacaccg gaaactcttc
     2701 acatttggcg tcaccttcag cactttcctt ctcgccctgt tctgcataac caagggtgga
     2761 atttacgtct tgaccctcct ggacaccttt gctgcgggca cctccatcct ttttgctgtc
     2821 ctcatggaag ccatcggagt ttcctggttt tatggagtgg acaggttcag caacgacatc
     2881 cagcagatga tggggttcag gccgggtcta tactggagac tgtgctggaa gttcgtcagt
     2941 cctgccttcc tcctgttcgt ggttgtggtc agcatcatca acttcaagcc actcacctac
     3001 gacgactaca tcttcccgcc ctgggccaac tgggtggggt ggggcatcgc cctgtcctcc
     3061 atggtcctgg tgcccatcta cgtcatctat aagttcctca gcacgcaggg ctctctttgg
     3121 gagagactgg cctatggcat cacgccagag aacgagcacc acctggtggc tcagagggac
     3181 atcagacagt tccagttgca acactggctg gccatctgag cctgcctgga ggagaaggag
     3241 gaacctctag agggcccggg atccaccgga tctagataac tgatcataat cagccatacc
     3301 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
     3361 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
     3421 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     3481 ggtttgtcca aactcatcaa tgtatcttaa cgcgtaaatt gtaagcgtta atattttgtt
     3541 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     3601 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     3661 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     3721 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     3781 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     3841 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     3901 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     3961 acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     4021 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     4081 ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag
     4141 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     4201 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     4261 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     4321 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     4381 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     4441 ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat
     4501 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     4561 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     4621 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga
     4681 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     4741 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     4801 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     4861 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     4921 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     4981 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga
     5041 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     5101 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     5161 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     5221 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     5281 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     5341 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     5401 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     5461 cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg
     5521 acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg
     5581 gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac
     5641 gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc
     5701 gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag
     5761 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     5821 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     5881 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     5941 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     6001 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     6061 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     6121 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     6181 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     6241 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     6301 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     6361 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     6421 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     6481 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     6541 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat
//
