LOCUS       sequence-311917-        7778 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For targeted insertion of V5-HA-2A-Luciferase reporter before the 
            stop codon of the human SLC12A5 gene that encodes neuronal chloride 
            transporter KCC2 protein..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7778)
  AUTHORS   Tang X, Drotar J, Li K, Clairmont CD, Brumm AS, Sullins AJ, Wu H, 
            Liu XS, Wang J, Gray NS, Sur M, Jaenisch R
  TITLE     Pharmacological enhancement of KCC2 gene expression exerts 
            therapeutic effects on human Rett syndrome neurons and Mecp2 mutant 
            mice.
  JOURNAL   Sci Transl Med. 2019 Jul 31;11(503). pii: 11/503/eaau0164. doi: 
            10.1126/scitranslmed.aau0164.
  PUBMED    31366578
REFERENCE   2  (bases 1 to 7778)
  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: "Sci Transl 
            Med."; date: "2019-07-31"; volume: "11(503). pii: 11/503/eaau0164. 
            doi"; pages: " 10.1126/scitranslmed.aau0164"
COMMENT     Sequence Label: human SLC12A5-V5-HA-2A-Luciferase repair template
FEATURES             Location/Qualifiers
     source          1..7778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    89..110
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(125..130,131..148,149..155)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    163..179
                     /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)."
     primer_bind     168..190
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     187..203
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     187..203
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1283..1324
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1325..1351
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1352..1408
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1409..3061
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     primer_bind     complement(1475..1493)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     primer_bind     complement(4193..4212)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4194..4212)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4219..4236)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4219..4235)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4228..4250)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4376..4804
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4463..4482)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4673..4694
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             5148..5942
                     /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(5202..5221)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5812..5831
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(5960..6028,6029..6820)
                     /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     complement(6178..6197)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6991..7579
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7480..7499
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7733..7750
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
       61 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
      121 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
      181 ttcacacagg aaacagctat gaccatgatt acgccaagct tggtaccgag ctcggatcca
      241 ctagtcctgg gggctaaggg ctgggggctg gggtgagcta aagggtcttg ctccccatgg
      301 cagagcaaga gagcagaagg catcctggtc tgtcagcctg cagacccagc tcagacattg
      361 tcttggtttc tccatctgag gacttagggg gttgggcccc attgctaagt ctcagaattc
      421 ccagttgcag gcagcggaaa ggtgaagggt gtgggggctg gcagagcagg actcagggca
      481 gatgaccatg gcccaagccc ccagtgtctt cctctttttc tgactctgct ctttttcttt
      541 ctctctttgc atctcttgtg tttcctgaag ggagtgggag aacttgtaag tgcttcagca
      601 ttttttcatt ctctctccta ggatggccag ggtccctacc ctcctcactc tgttgtgaac
      661 ccctaattgg tgccacgacc tctgggatct ctgaatagcc tagcctggag atgtttagga
      721 ttggtggtcc taggcttgca agaaccagtc cccagcagcc cagttcgggc tggaagggcg
      781 actggctcca atcttctcta cccccccggc tcacgcggtc tccactcctc cttcctgccg
      841 caggaaccag tccaacgtgc ggcgcatgca cacggccgtg cggctgaacg aggtcatcgt
      901 gaagaaatcc cgggacgcca agcttgtttt gctcaacatg cctgggcctc cccgcaaccg
      961 caatggtgat gaaaactgta tcctggaatt aaaattgggg gaaagaggga ggtggacgtc
     1021 agggaatctg ggtcctgtcc ctgggatgga agagctgagc tgttcctgcc tccggatcag
     1081 cacctcggac agggacacgg gcgcgagagg tcccctggca gccgagcgcg accccaattt
     1141 cgtcgggagg gaaggagacc gggtctttct ccttgaccgg ctctcagaca tggagtttct
     1201 cgaggtcctc acagagcacc tggaccgggt gatgctggtc cgcggcggcg gccgcgaggt
     1261 catcaccatc tactccgcta gcggcaaacc gattccgaat ccgctgctgg gcctggattc
     1321 cacctaccca tacgatgttc cagattacgc tgccactaac ttctccctgt tgaaacaagc
     1381 aggggatgtc gaagagaatc ccgggccaat ggaagacgcc aaaaacataa agaaaggccc
     1441 ggcgccattc tatccgctgg aagatggaac cgctggagag caactgcata aggctatgaa
     1501 gagatacgcc ctggttcctg gaacaattgc ttttacagat gcacatatcg aggtggacat
     1561 cacttacgct gagtacttcg aaatgtccgt tcggttggca gaagctatga aacgatatgg
     1621 gctgaataca aatcacagaa tcgtcgtatg cagtgaaaac tctcttcaat tctttatgcc
     1681 ggtgttgggc gcgttattta tcggagttgc agttgcgccc gcgaacgaca tttataatga
     1741 acgtgaattg ctcaacagta tgggcatttc gcagcctacc gtggtgttcg tttccaaaaa
     1801 ggggttgcaa aaaattttga acgtgcaaaa aaagctccca atcatccaaa aaattattat
     1861 catggattct aaaacggatt accagggatt tcagtcgatg tacacgttcg tcacatctca
     1921 tctacctccc ggttttaatg aatacgattt tgtgccagag tccttcgata gggacaagac
     1981 aattgcactg atcatgaact cctctggatc tactggtctg cctaaaggtg tcgctctgcc
     2041 tcatagaact gcctgcgtga gattctcgca tgccagagat cctatttttg gcaatcaaat
     2101 cattccggat actgcgattt taagtgttgt tccattccat cacggttttg gaatgtttac
     2161 tacactcgga tatttgatat gtggatttcg agtcgtctta atgtatagat ttgaagaaga
     2221 gctgtttctg aggagccttc aggattacaa gattcaaagt gcgctgctgg tgccaaccct
     2281 attctccttc ttcgccaaaa gcactctgat tgacaaatac gatttatcta atttacacga
     2341 aattgcttct ggtggcgctc ccctctctaa ggaagtcggg gaagcggttg ccaagaggtt
     2401 ccatctgcca ggtatcaggc aaggatatgg gctcactgag actacatcag ctattctgat
     2461 tacacccgag ggggatgata aaccgggcgc ggtcggtaaa gttgttccat tttttgaagc
     2521 gaaggttgtg gatctggata ccgggaaaac gctgggcgtt aatcaaagag gcgaactgtg
     2581 tgtgagaggt cctatgatta tgtccggtta tgtaaacaat ccggaagcga ccaacgcctt
     2641 gattgacaag gatggatggc tacattctgg agacatagct tactgggacg aagacgaaca
     2701 cttcttcatc gttgaccgcc tgaagtctct gattaagtac aaaggctatc aggtggctcc
     2761 cgctgaattg gaatccatct tgctccaaca ccccaacatc ttcgacgcag gtgtcgcagg
     2821 tcttcccgac gatgacgccg gtgaacttcc cgccgccgtt gttgttttgg agcacggaaa
     2881 gacgatgacg gaaaaagaga tcgtggatta cgtcgccagt caagtaacaa ccgcgaaaaa
     2941 gttgcgcgga ggagttgtgt ttgtggacga agtaccgaaa ggtcttaccg gaaaactcga
     3001 cgcaagaaaa atcagagaga tcctcataaa ggccaagaag ggcggaaaga tcgccgtgta
     3061 attaattaat gagaaccagg acctgccacc cgggcccgag cgcgcccggc ccgcggctcc
     3121 ggagccctcg ccgcgccccc cgccgctgtc accgtttaca tacagaccct gtgcccgtgt
     3181 cctggcccct taccccgctg cctgaagccc ggaggccacg cctgttgggg ctgattcgga
     3241 gagggcgccc cgccgcgcag agaccagagc tcctcagtgc cagtttggcc cctgggtctt
     3301 cgctgccctt tttctaagcc cggcctcgtc tcgccggagg agacgctgca ataaaggttg
     3361 ggagaaggcg cggaaaggag aggagctggg gccttgggga cccccaggta gtccatgcgg
     3421 cccattcctc cccttcccac tcccgccgcg gtcctcgctc tgcgctcctc cggcgctgct
     3481 ccctggctcc cggcggcccg gaggcccgcg gggtgggaag gccgcgcttg ccgtctccgc
     3541 cgccccttct cgccgagccg tggggcgcgg gcggccgagc ctatacatag tgtacaggag
     3601 acatcgcgtg tatttttaac gtccccatat ttatgtgact agaagcgcaa cagacttctc
     3661 gccatagtcg agctctcccg ctgggggcac tgcggggagg cgaggcctcg ggaagctgaa
     3721 ttttccttga cgtccaagag tttgagagcg aaagtgcttt aggcccaggc gggggtcgtg
     3781 gcctcgttcc ctcgacacct ccgtcctgct ctcgcctctt cgccctttcc gcgcgccctt
     3841 ggcttcccac cctcctctcc agtccttttc cgagatgagg tgagacaagg gtccaacttt
     3901 tcctggattc gcctcccagc ggacgtgagc ttccactgcg gctgcagaga cgcgagcaac
     3961 ctcttctcat cggctcttat gcaagttggg gccaggatag gggaggggtg ctcctcaaga
     4021 ggaagaaacc gagaggcccg cgccccaccg aggaagcccc gccccggtgc cttcgctggg
     4081 gagcaggcgt ctctcctcag tcggcttgtc gcggccggcc aataagggcg aattctgcag
     4141 atatccatca cactggcggc cgctcgagca tgcatctaga gggcccaatt cgccctatag
     4201 tgagtcgtat tacaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     4261 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     4321 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggacgcg
     4381 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     4441 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     4501 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     4561 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     4621 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     4681 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     4741 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     4801 aattttaaca aaattcaggg cgcaagggct gctaaaggaa gcggaacacg tagaaagcca
     4861 gtccgcagaa acggtgctga ccccggatga atgtcagcta ctgggctatc tggacaaggg
     4921 aaaacgcaag cgcaaagaga aagcaggtag cttgcagtgg gcttacatgg cgatagctag
     4981 actgggcggt tttatggaca gcaagcgaac cggaattgcc agctggggcg ccctctggta
     5041 aggttgggaa gccctgcaaa gtaaactgga tggctttctt gccgccaagg atctgatggc
     5101 gcaggggatc aagatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     5161 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     5221 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     5281 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     5341 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     5401 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     5461 cccaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     5521 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     5581 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     5641 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     5701 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     5761 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     5821 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     5881 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     5941 gaattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     6001 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     6061 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     6121 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     6181 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     6241 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     6301 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     6361 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     6421 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     6481 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     6541 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     6601 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     6661 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     6721 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     6781 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     6841 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     6901 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     6961 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     7021 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     7081 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     7141 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     7201 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     7261 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     7321 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     7381 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     7441 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     7501 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     7561 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     7621 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     7681 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     7741 agtgagcgag gaagcggaag agcgcccaat acgcaaac
//
