LOCUS       sequence-199373-        8156 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses rat KCC2 (b isoform) with eGFP tag at the N-terminus of 
            KCC2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8156)
  AUTHORS   Pellegrino C, Gubkina O, Schaefer M, Becq H, Ludwig A, Mukhtarov M, 
            Chudotvorova I, Corby S, Salyha Y, Salozhin S, Bregestovski P, 
            Medina I
  TITLE     Knocking down of the KCC2 in rat hippocampal neurons increases 
            intracellular chloride concentration and compromises neuronal 
            survival.
  JOURNAL   J Physiol. 2011 May 15;589(Pt 10):2475-96. doi: 
            10.1113/jphysiol.2010.203703. Epub 2011 Mar 21.
  PUBMED    21486764
REFERENCE   2  (bases 1 to 8156)
  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: "J 
            Physiol."; date: "2011-05-15"; volume: "589(Pt 10):2475-96. doi"; 
            pages: " 10.1113/jphysiol.2010.203703. Epub 2011 Mar 21"
COMMENT     Sequence Label: eGFP-KCC2
FEATURES             Location/Qualifiers
     source          1..8156
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      547..556
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(553..555,556..558,559..1269)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(553..555,556..558,559..1269)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(598..619)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(859..878)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1206..1227
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             complement(2203..2214)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4884..5005
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4921..4940)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4975..4994
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5012..5467)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5149..5170)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5361..5380
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5494..5598
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5600..5957
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5600..5791
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5623..5643)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5808..5943
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5992..6786
                     /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             5992..6786
                     /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(6046..6065)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6656..6675
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6974..6993)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7018..7065
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7394..7982
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7883..7902
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      541 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      601 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      661 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      721 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      781 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      841 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      901 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
      961 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1021 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1081 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1141 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1201 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1261 ctgtacaagt ccggactcag atctcgagct caagcttcgg cacgaggcga aggcgggcag
     1321 aggggcgcgg gcgaagaggc gcagccatcc cgagcccggc gccgcgcagc caccatgctc
     1381 aacaacctga cggactgcga ggacggcgat gggggagcca acccgggtga cggcaatccc
     1441 aaggagagca gccccttcat caacagcacg gacacggaga aggggagaga gtatgatggc
     1501 aggaacatgg ccctgtttga ggaggagatg gacaccagcc ccatggtatc ctccctgctc
     1561 agtgggctgg ccaactacac caacctgcct cagggaagca aagagcacga agaagcagaa
     1621 aacaatgagg gcggaaagaa gaagccggtg caggccccac gcatgggcac cttcatgggc
     1681 gtgtacctcc cgtgcctgca gaacatcttt ggtgttatcc tctttctgcg gctcacttgg
     1741 gtggtgggaa tcgcaggcat catggagtcc ttctgcatgg tcttcatctg ctgctcctgc
     1801 acgatgctca cagccatttc catgagcgca attgcaacca atggtgttgt gcctgctggt
     1861 ggctcctact acatgatttc caggtctctg ggcccggagt ttgggggcgc cgtgggcctc
     1921 tgcttctacc tgggcactac ctttgctggg gctatgtaca tcctgggcac catcgagatc
     1981 ctgctggctt acctcttccc agcgatggcc atcttcaagg cagaagatgc cagtggggag
     2041 gcagccgcca tgttgaataa catgcgggtg tatggcacct gtgtgctcac ctgcatggcc
     2101 accgtagtct ttgtgggcgt caagtacgtg aacaagtttg ccctggtctt cctgggttgc
     2161 gtgatcctct ccatcctggc catctacgca ggggtcatca agtctgcctt cgatccaccc
     2221 aatttcccga tttgcctcct ggggaaccgc acgctgtctc gccatggctt tgatgtctgt
     2281 gccaagctgg cttgggaagg aaatgagaca gtgaccacac ggctctgggg cctattctgt
     2341 tcctcccgcc tcctcaatgc cacctgtgat gagtacttca cccgaaacaa tgtcacagag
     2401 atccagggca ttcctggtgc tgcaagtggc ctcatcaaag agaacctgtg gagttcctac
     2461 ctgaccaagg gggtgatcgt ggagaggcgt gggatgccct ctgtgggcct ggcagatggt
     2521 acccccgttg acatggacca cccctatgtc ttcagtgata tgacctccta cttcaccctg
     2581 cttgttggca tctatttccc ctcagtcaca gggatcatgg ctggctcgaa ccggtccgga
     2641 gacctgcggg atgcccagaa gtctatccct actggaacta tcttggccat tgctacgacc
     2701 tctgctgtct acatcagctc tgttgttctg ttcggagcct gcatcgaagg ggtcgtccta
     2761 cgggacaagt ttggggaagc tgtgaatggc aatctggtgg tgggcaccct ggcctggcct
     2821 tctccttggg tcattgtcat aggctctttc ttctctacct gcggagctgg actacagagc
     2881 ctcacagggg ccccacgcct gctgcaggcc atctcccggg atggcatagt gcccttcctg
     2941 caggtctttg gccatggcaa agccaacgga gagccaacct gggcgctgct gctgactgcc
     3001 tgcatctgtg agatcggcat cctcatcgcc tccctggatg aggtcgcccc tatcctttcc
     3061 atgttcttcc tgatgtgtta catgtttgtg aacttggctt gcgcggtgca gacactgctg
     3121 aggacgccca actggaggcc acgcttccga tattaccact ggaccctctc cttcctgggc
     3181 atgagcctct gcctggccct gatgttcatt tgctcctggt attatgcgct ggtagctatg
     3241 ctcatcgctg gcctcatcta taagtacatc gagtaccggg gggcagagaa ggagtggggg
     3301 gatgggatcc gaggcctgtc tctcagtgca gctcgctatg ctctcttgcg tctggaggaa
     3361 ggacccccgc atacaaagaa ctggaggccc cagctactgg tgctggtgcg tgtggaccag
     3421 gaccagaacg tggtgcaccc gcagctgctg tccttgacct cccagctcaa ggcagggaag
     3481 ggcctgacca ttgtgggctc tgtccttgag ggcacctttc tggacaacca ccctcaggct
     3541 cagcgggcag aggagtctat ccggcgcctg atggaggctg agaaggtgaa gggcttctgc
     3601 caggtagtga tctcctccaa cctgcgtgac ggtgtgtccc acctgatcca atccgggggc
     3661 ctcgggggcc tgcaacacaa cactgtgcta gtgggctggc ctcgcaactg gcgacagaag
     3721 gaggatcatc agacatggag gaacttcatc gaactcgtcc gggaaactac agctggccac
     3781 ctcgccctgc tggtcaccaa gaatgtttcc atgttccccg ggaaccctga gcgtttctct
     3841 gagggcagca ttgacgtgtg gtggatcgtg cacgacgggg gcatgctcat gctgttgccc
     3901 ttcctcctgc gtcaccacaa ggtctggagg aaatgcaaaa tgcggatctt caccgtggcg
     3961 cagatggatg acaacagcat tcagatgaag aaagacctga ccacgtttct gtaccactta
     4021 cgaattactg cagaggtgga agtcgtggag atgcacgaga gcgacatctc agcatacacc
     4081 tacgagaaga cattggtaat ggaacaacgt tctcagatcc tcaaacagat gcacctcacc
     4141 aagaacgagc gggaacggga gatccagagc atcacagatg aatctcgggg ctccattcgg
     4201 aggaagaatc cagccaacac tcggctccgc ctcaatgttc ccgaagagac agcttgtgac
     4261 aacgaggaga agccagaaga ggaggtgcag ctgatccatg accagagtgc tcccagctgc
     4321 cctagcagct cgccgtctcc aggggaggag cctgaggggg agggggagac agacccagag
     4381 aaggtgcatc tcacctggac caaggataag tcagcggctc agaagaacaa aggccccagt
     4441 cccgtctcct cggaggggat caaggacttc ttcagcatga agccggagtg ggaaaacttg
     4501 aaccagtcca acgtgcggcg catgcacaca gctgtgcggc tgaacgaggt catcgtgaat
     4561 aaatcccggg atgccaagtt ggtgttgctc aacatgcccg ggcctccccg caaccgcaat
     4621 ggagatgaaa actacatgga gttcctggag gtcctcactg agcaactgga ccgggtgatg
     4681 ctggtccgcg gtggtggccg agaggtcatc accatctact cctgaattct gcagtcgacg
     4741 gtaccgcggg cccgggatcc accggatcta gataactgat cataatcagc cataccacat
     4801 ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata
     4861 aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
     4921 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4981 tgtccaaact catcaatgta tcttaacgcg taaattgtaa gcgttaatat tttgttaaaa
     5041 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     5101 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     5161 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     5221 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     5281 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     5341 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     5401 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     5461 ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     5521 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     5581 tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag
     5641 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     5701 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     5761 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     5821 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     5881 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     5941 gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa
     6001 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     6061 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     6121 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag
     6181 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     6241 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     6301 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     6361 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     6421 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     6481 gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat
     6541 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     6601 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     6661 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     6721 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     6781 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     6841 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     6901 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta
     6961 gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg
     7021 caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc
     7081 ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc
     7141 gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag
     7201 ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg
     7261 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     7321 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     7381 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     7441 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     7501 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     7561 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     7621 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     7681 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     7741 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     7801 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     7861 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     7921 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     7981 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     8041 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcattagt
     8101 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccg
//
