LOCUS       sequence-63774-e        6499 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses hM4D (Gi) neuronal inhibitor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6499)
  AUTHORS   Armbruster BN, Li X, Pausch MH, Herlitze S, Roth BL
  TITLE     Evolving the lock to fit the key to create a family of G 
            protein-coupled receptors potently activated by an inert ligand.
  JOURNAL   Proc Natl Acad Sci U S A. 2007 Mar 20;104(12):5163-8. Epub 2007 Mar 
            2.
  PUBMED    17360345
REFERENCE   2  (bases 1 to 6499)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2007-03-20"; volume: "104(12)"; pages: 
            "5163-8. Epub 2007 Mar 2"
COMMENT     Sequence Label: pcDNA5/FRT-HA-hM4D(Gi)
FEATURES             Location/Qualifiers
     source          1..6499
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             941..967
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2452..2469)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2458..2682
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2815..2834)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2965..3012
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3020..4042
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3020..4042
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4172..4293
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4209..4228)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4263..4282
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4342..4358)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4342..4358)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4355..4377)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4366..4382
                     /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(4390..4396,4397..4414,4415..4420))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4435..4456
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4573..4590)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4744..5332)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4824..4843)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5503..6294,6295..6363))
                     /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     6126..6145
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6364..6468)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttggta ccgagctcgg atccaccatg tacccatacg atgttccaga
      961 ttacgctatg gccaacttca cacctgtcaa tggcagctcg ggcaatcagt ccgtgcgcct
     1021 ggtcacgtca tcatcccaca atcgctatga gacggtggaa atggtcttca ttgccacagt
     1081 gacaggctcc ctgagcctgg tgactgtcgt gggcaacatc ctggtgatgc tgtccatcaa
     1141 ggtcaacagg cagctgcaga cagtcaacaa ctacttcctc ttcagcctgg cgtgtgctga
     1201 tctcatcata ggcgccttct ccatgaacct ctacaccgtg tacatcatca agggctactg
     1261 gcccctgggc gccgtggtct gcgacctgtg gctggccctg gactgcgtgg tgagcaacgc
     1321 ctccgtcatg aaccttctca tcatcagctt tgaccgctac ttctgcgtca ccaagcctct
     1381 cacctaccct gcccggcgca ccaccaagat ggcaggcctc atgattgctg ctgcctgggt
     1441 actgtccttc gtgctctggg cgcctgccat cttgttctgg cagtttgtgg tgggtaagcg
     1501 gacggtgccc gacaaccagt gcttcatcca gttcctgtcc aacccagcag tgacctttgg
     1561 cacagccatt gctggcttct acctgcctgt ggtcatcatg acggtgctgt acatccacat
     1621 ctccctggcc agtcgcagcc gagtccacaa gcaccggccc gagggcccga aggagaagaa
     1681 agccaagacg ctggccttcc tcaagagccc actaatgaag cagagcgtca agaagccccc
     1741 gcccggggag gccgcccggg aggagctgcg caatggcaag ctggaggagg cccccccgcc
     1801 agcgctgcca ccgccaccgc gccccgtggc tgataaggac acttccaatg agtccagctc
     1861 aggcagtgcc acccagaaca ccaaggaacg cccagccaca gagctgtcca ccacagaggc
     1921 caccacgccc gccatgcccg cccctcccct gcagccgcgg gccctcaacc cagcctccag
     1981 atggtccaag atccagattg tgacgaagca gacaggcaat gagtgtgtga cagccattga
     2041 gattgtgcct gccacgccgg ctggcatgcg ccctgcggcc aacgtggccc gcaagttcgc
     2101 cagcatcgct cgcaaccagg tgcgcaagaa gcggcagatg gcggcccggg agcgcaaagt
     2161 gacacgaacg atctttgcca ttctgctagc cttcatcctc acctggacgc cctacaacgt
     2221 catggtcctg gtgaacacct tctgccagag ctgcatccct gacacggtgt ggtccattgg
     2281 ctactggctc tgctacgtca acagcaccat caaccctgcc tgctatgctc tgtgcaacgc
     2341 cacctttaaa aagaccttcc ggcacctgct gctgtgccag tatcggaaca tcggcactgc
     2401 caggtaggcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg
     2461 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     2521 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     2581 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     2641 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     2701 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     2761 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     2821 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     2881 gggggtccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2941 ttagggtgat ggttcacgta cctagaagtt cctattccga agttcctatt ctctagaaag
     3001 tataggaact tccttggcca aaaagcctga actcaccgcg acgtctgtcg agaagtttct
     3061 gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg
     3121 tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga
     3181 tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc
     3241 ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct cccgccgtgc
     3301 acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt
     3361 cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc
     3421 attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc
     3481 tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc
     3541 gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt
     3601 gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat
     3661 tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg
     3721 gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga
     3781 gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta
     3841 tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc
     3901 aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc
     3961 cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac
     4021 tcgtccgagg gcaaaggaat agcacgtact acgagatttc gattccaccg ccgccttcta
     4081 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     4141 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     4201 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     4261 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     4321 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     4381 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     4441 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4501 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4561 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4621 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4681 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4741 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4801 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4861 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4921 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4981 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5041 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5101 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5161 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     5221 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5281 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5341 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5401 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5461 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     5521 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5581 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     5641 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5701 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5761 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5821 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5881 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5941 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6001 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6061 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     6121 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6181 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6241 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6301 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6361 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     6421 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6481 aaaagtgcca cctgacgtc
//
