LOCUS       sequence-427235-        7840 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Mammalian expression of improved GABA sensor (negative change in 
            fluorescence).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7840)
  AUTHORS   Kolb I, Hasseman JP, Matsumoto A, Arthur BJ, Zhang Y, Tsang A, Reep 
            D, Tsegaye G, Zheng J, Patel R, Looger LL, Marvin JS, Korff WL, 
            Yonehara K, Turner GC
  TITLE     iGABASnFR2: Improved genetically encoded protein sensors of GABA
  JOURNAL   bioRxiv 2025.03.25.644953
REFERENCE   2  (bases 1 to 7840)
  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 
            2025.03.25.644953"
COMMENT     Sequence Label: pGP-CAG-iGABASnFR2n-WPRE-bGH-polyA
FEATURES             Location/Qualifiers
     source          1..7840
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1594..1616
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1678..1697
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1752..1761
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     sig_peptide     1758..1820
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTGD"
     primer_bind     2864..2885
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2988..3009)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             3516..3545
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3549..3695
                     /codon_start=1
                     /product="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /label=PDGFR-beta TM domain
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
     intron          3764..3896
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3879..3898
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    3905..4493
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3958..3978)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4376..4387)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4520..4537)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4526..4637
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4864..4883)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4929..4984
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4983..5002)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5343..5359)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5356..5378)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5367..5383
                     /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(5391..5397,5398..5415,5416..5421))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5436..5457
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5516..5711
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5562..5697
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5717..5851
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5754..5773)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5808..5827
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5919..5936)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6090..6678)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6170..6189)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6849..7640,7641..7709))
                     /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     7472..7491
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7710..7814)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg ggcgagggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
     1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     1681 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcggtacc gcgggcccgg
     1741 gaaccggtgc cgccaccatg gagacagaca cactcctgct atgggtactg ctgctctggg
     1801 ttccaggttc cactggtgac agatctgaaa gcatcaattt cgtgagctgg ggcggtagca
     1861 cccaggatgc gcagaagcag gcctgggccg acccgttcag caaggccagc ggcattaccg
     1921 tggtccagga tgggcccacc gactacggca aactcaaggc catggtcgaa agcggcaacg
     1981 tgcagtggga cgtggtggat gtagaggccg acttcgcctt gcgcgccgcc gccgaaggcc
     2041 tgctcgaacc cctggatttc tcggtgatcc agcgcgacaa gatcgatccg cggttcgtga
     2101 gtgaccatgg agtgggggcc ttcctgttct ctcacgtgct cggctacaac gaaggcaagc
     2161 tcggagccag caagccccag gactggaccg cgctgttcga caccaagacc taccccggca
     2221 aacgcgccct ctacaaatgg ccgagccctg gcgtgctcga actggccctg ctcgccgacg
     2281 gcgtaccggc cgacaagctc tacccgctgg acctggaccg cgccttcaag aaactcgaca
     2341 ccatcaagaa agacatcgtc tggtggggcg gcggtgcaca gtcgcagcag ctgctggcct
     2401 ccggcgaagt cagcatgggc cagttctgga acggtcgcat ccacgccctg caagaagacg
     2461 gcgcgccagt gggcgtgagc tggaagcaga acctggtgat ggccgacatc ctcgtcgtgc
     2521 ctaaaggcac gaagaacaaa gccgccgcga tgaagttcct ggccagtgcc agcagcgcca
     2581 aaggccagga cgacttttcc gccctgaccg cctatgctcc agtgaacatc gacagtgtgc
     2641 agcgcctcga ccttgcgcag gtccggatca ccgccgacaa gcagaagaac ggcatcatgg
     2701 cgaacttcaa gatcccgcac aacgtggagg acggcagcgt gcagctcgcc gaccactacc
     2761 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     2821 cccagtccgt gctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     2881 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagggc ggtaccggag
     2941 ggagcatgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     3001 acggcgacgt aaacggccac aagttcagcg tgcgcggcga gggcgagggc gatgccacca
     3061 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     3121 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     3181 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatca
     3241 gcttcaagga cgacggcacc tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     3301 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     3361 acaagctgga gtacaactgg aacgccaacc tggcaccgaa cctgccgacc gcctacgtca
     3421 aggatcagat caccctcgat ttcgcctact gggccaagaa cggtccggcc attgcgacac
     3481 ggtggaatga atggctagtc aaactgcagg tcgacgaaca aaaactcatc tcagaagagg
     3541 atctgaatgc tgtgggccag gacacgcagg aggtcatcgt ggtgccacac tccttgccct
     3601 ttaaggtggt ggtgatctca gccatcctgg ccctggtggt gctcaccatc atctccctta
     3661 tcatcctcat catgctttgg cagaagaagc cacgttagga attcgatatc ggtaccgatg
     3721 aatagctcga ggtaccgatg aatagctaag gtcgaggccg caggtaagta tcaaggttac
     3781 aagacaggtt taaggagacc aatagaaact gggcttgtcg agacagagaa gactcttgcg
     3841 tttctgatag gcacctattg gtcttactga catccacttt gcctttctct ccacaggtgt
     3901 cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     3961 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     4021 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     4081 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     4141 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     4201 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     4261 gctgttgggc actgacaatt ccgtggtgtt gtcggggaag ctgacgtcct ttccatggct
     4321 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     4381 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     4441 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcctggaat
     4501 tcgagctcgg tacgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     4561 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     4621 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     4681 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggccagcttt tgttcccttt
     4741 agggttaatt gcgcgcttgg cgctaatcat ggtcatagct gtttccactc ctcaggtgca
     4801 ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca aataccactg
     4861 agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg agcatctgac
     4921 ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct
     4981 ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg agtatttggt
     5041 ttagagtttg gcaacatatg ccatatgctg gctgccatga acaaaggtgg ctataaagag
     5101 gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga aaagccttga
     5161 cttgaggtta gatttttttt tatattttgt ttgtgttatt tttttcttta acatccctaa
     5221 aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta ctcccagtca
     5281 tagctgtccc tcttctctta tgaagatccc tcgacctgca gcccaagctt ggcgtaatca
     5341 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     5401 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     5461 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc
     5521 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     5581 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     5641 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     5701 gcttttgcaa aaagctaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     5761 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     5821 actcatcaat gtatcttatc atgtctggat ccgctgcatt aatgaatcgg ccaacgcgcg
     5881 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     5941 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     6001 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     6061 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     6121 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     6181 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     6241 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg
     6301 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     6361 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     6421 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     6481 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     6541 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6601 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     6661 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     6721 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6781 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     6841 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     6901 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     6961 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     7021 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     7081 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     7141 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     7201 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     7261 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     7321 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     7381 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     7441 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     7501 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     7561 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     7621 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     7681 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     7741 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     7801 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
//
