LOCUS       sequence-275780-        7454 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Cre-dependent expression of PSAM-GlyR (L141F,Y115F) neuronal 
            inhibitor, with IRES-EGFP marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7454)
  AUTHORS   Magnus CJ, Lee PH, Atasoy D, Su HH, Looger LL, Sternson SM
  TITLE     Chemical and genetic engineering of selective ion channel-ligand 
            interactions.
  JOURNAL   Science. 2011 Sep 2;333(6047):1292-6.
  PUBMED    21885782
REFERENCE   2  (bases 1 to 7454)
  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: "Science."; 
            date: "2011-09-2"; volume: "333(6047)"; pages: "1292-6"
COMMENT     Sequence Label: rAAV-syn::FLEX-rev::PSAML141F,Y115F:GlyR-IRES-GFP
FEATURES             Location/Qualifiers
     source          1..7454
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   55..184
                     /label=AAV2 ITR
     intron          777..909
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     892..911
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     protein_bind    922..955
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    1018..1051
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(1062..1775,1776..1778))
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1107..1128)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1456..1475
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1715..1736
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     misc_feature    1802..2353
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     2192..2209
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     protein_bind    complement(3708..3741)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(3812..3845)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3917..4505
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3970..3990)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4388..4399)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4583..4704
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4594..4613)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4648..4667
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4866..4995
                     /label=AAV2 ITR
     rep_origin      complement(5232..5820)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5312..5331)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5991..6782,6783..6851))
                     /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     6614..6633
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6852..6956)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6982..7437)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7119..7140)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7331..7350
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctgcgc
       61 gctcgctcgc tcactgaggc cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc
      121 ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt
      181 tccttgtagt taatgattaa cccgccatgc tacttatcta cgtagccatg ctctaggaag
      241 atccgagctc cagtgtgctg gaattcgccc ttttcagtat ttaaattgac gaccgacccc
      301 gacccactgg acaagcaccc aacccccatt ccccaaattg cgcatcccct atcagagagg
      361 gggaggggaa acaggatgcg gcgaggcgcg tgcgcactgc cagcttcagc accgcggaca
      421 gtgccttcgc ccccgcctgg cggcgcgcgc caccgccgcc tcagcactga aggcgcgctg
      481 acgtcactcg ccggtccccc gcaaactccc cttcccggcc accttggtcg cgtccgcgcc
      541 gccgccggcc cagccggacc gcaccacgcg aggcgcgaga taggggggca cgggcgcgac
      601 catctgcgct gcggcgccgg cgactcagcg ctgcctcagt ctgcggtggg cagcggagga
      661 gtcgtgtcgt gcctgagagc gcagctgtgc tcctgggcac cgcgcagtcc gcccccgcgg
      721 ctcctggcca gaccacccct aggaccccct gccccaagtc gcagccgttg gatcaggtaa
      781 gtatcaaggt tacaagacag gtttaaggag accaatagaa actgggcttg tcgagacaga
      841 gaagactctt gcgtttctga taggcaccta ttggtcttac tgacatccac tttgcctttc
      901 tctccacagg tgatgatccc cataacttcg tataaagtat cctatacgaa gttatatcaa
      961 aataggaaga ccaatgcttc accatcgacc cgaattgcca agcatcacca tcgacacata
     1021 acttcgtata atgtatgcta tacgaagtta tactagctag attacttgta cagctcgtcc
     1081 atgccgagag tgatcccggc ggcggtcacg aactccagca ggaccatgtg atcgcgcttc
     1141 tcgttggggt ctttgctcag cttggactgg gtgctcaggt agtggttgtc gggcagcagc
     1201 acggggccgt cgccgatggg ggtgttctgc tggtagtggt cggcgagctg cacgctgccg
     1261 tcctcgatgt tgtggcggat cttgaagttc accttgatgc cgttcttctg cttgtcggcc
     1321 atgatataga cgttgtggct gttgtagttg tactccagct tgtgccccag gatgttgccg
     1381 tcctccttga agtcgatgcc cttcagctcg atgcggttca ccagggtgtc gccctcgaac
     1441 ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga agaagatggt gcgctcctgg
     1501 acgtagcctt cgggcatggc ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag
     1561 cggctgaagc actgcacgcc gtaggtcagg gtggtcacga gggtgggcca gggcacgggc
     1621 agcttgccgg tggtgcagat gaacttcagg gtcagcttgc cgtaggtggc atcgccctcg
     1681 ccctcgccgg acacgctgaa cttgtggccg tttacgtcgc cgtccagctc gaccaggatg
     1741 ggcaccaccc cggtgaacag ctcctcgccc ttgctcaccg gatcccccat ggttgtggcc
     1801 atattatcat cgtgtttttc aaaggaaaac cacgtccccg tggttcgggg ggcctagacg
     1861 tttttttaac ctcgactaaa cacatgtaaa gcatgtgcac cgaggcccca gatcagatcc
     1921 catacaatgg ggtaccttct gggcatcctt cagccccttg ttgaatacgc ttaggagagc
     1981 catttgactc tttccacaac tatccaactc acaacgtggc actggggttg tgccgccttt
     2041 gcaggtgtat cttatacacg tggcttttgg ccgcagaggc acctgtcgcc aggtgggggg
     2101 ttccgctgcc tgcaaagggt cgctacagac gttgtttgtc ttcaagaagc ttccagagga
     2161 actgcttcct tcacgacatt caacagacct tgcattcctt tggcgagagg ggaaagaccc
     2221 ctaggaatgc tcgtcaagaa gacagggcca ggtttccggg ccctcacatt gccaaaagac
     2281 ggcaatatgg tggaaaataa catatagaca aacgcacacc ggccttattc caagcggctt
     2341 cggccagtaa cgttaggggg gggggagggg ggaattgcgg ccgcttactg gttgtgtacg
     2401 tcctcccttc gcacaatctt gtagatgatc caataaaaca tgttgaaaat caggaaagcc
     2461 atggggaatc cgatccggga gattttgtcg atctttttcg ctctctgaat aaagagtttg
     2521 cgcatttcct ccgggctctt agatggagca gggggtgggt tggttgtgtt gctattgttg
     2581 gctcccttca cggaaattcc gtctttagcc tggaggcaag caggtcccat accataggcg
     2641 ctaaagttga agcgtccttc tcctgcctca tcctctttgt ggtggcgtcg ttttcttctg
     2701 aaccgcaaca gctctttatg ctgtcgagac acaaaattga cagcggcgta ttccagcagt
     2761 gcgctgaaga caaacagaag gcagacagcc atccagatat cgatggcctt cacgtaagaa
     2821 accttgggca agctggctcg gctgcctgag ctctgagtag tcattgtcag cactgtggtg
     2881 atgcccagtc cgaccctagc tggagcggcg tccatattaa tccagaagga gatccatgac
     2941 aaaatcacta taagcaagct tgggatatac atttggatca gataataacc catgcgtctc
     3001 cgcatggtca ctgtgaaggt gacatctggg tagggctctt tgcagcattc ataaaatcgc
     3061 tcgcttcttt taccggggat tcccacaagg tcccactccc cattcgggat gtaaccagag
     3121 atgtcagcct cctgcatttg cagatccagg ctccatccgc cgtagctcca gctcccgaat
     3181 ttgagtttgc agtgctggac gtcgaaggga aaccagcgga cgtcgatata gcatgaggac
     3241 ttgaagattc caggggggaa gtactggcag tggcctgaag aattgaccaa cacgttggtg
     3301 tgaaatgttg catcaaacct ttcgtctgcg ctgttgaaca gcaggatatc tggtttccag
     3361 atttggccgt cagggaatct tacagtcttt acacctgggt attcgctcac attccactgc
     3421 aagtagtgat cggtccagga catctgcaac caaatattag tggtcagcac ctggttcttt
     3481 tcatccacgt ccatgatctg cagcaaggag aggctgaagt acactgtcag tggctgggaa
     3541 tcatttgcga ccggccgctc cagggggtta taattcttaa caagctcctt atacagtttg
     3601 cgctggaact caccctgcag gctaacgtgc agaagggaag cagccagggc gagccacacg
     3661 ccgcctggag aacagcgcat ggtgaattca agcttgctag ccctagtata acttcgtata
     3721 ggatacttta tacgaagtta tcattgggat tcttcctatt ttgatccaag catcaccatc
     3781 gaccctctag tccagatctc accatcgacc cataacttcg tatagcatac attatacgaa
     3841 gttatgtccc tcgaagaggt tcgaaacact gaaaagggcg aattctgcag atgatccact
     3901 cgagaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
     3961 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca
     4021 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc
     4081 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc
     4141 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt
     4201 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
     4261 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc
     4321 ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta
     4381 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg
     4441 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc
     4501 cccgcatcga taccgtcgac ccgggcggcc gctcgagcat gcatctagag tcgacccggg
     4561 cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga
     4621 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     4681 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     4741 cagggggaga tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc
     4801 gataaggatc ttcctagagc atggctacgt agataagtag catggcgggt taatcattaa
     4861 ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     4921 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     4981 cgagcgagcg cgcagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5041 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5101 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5161 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5221 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5281 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5341 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5401 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     5461 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5521 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5581 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5641 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     5701 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5761 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     5821 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     5881 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     5941 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6001 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6061 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6121 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6181 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6241 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6301 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6361 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6421 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6481 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6541 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6601 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6661 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6721 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6781 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     6841 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     6901 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     6961 tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     7021 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     7081 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     7141 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     7201 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     7261 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     7321 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     7381 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc
     7441 attcgccatt cagg
//
