LOCUS       sequence-353882-        5710 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express human melanocortin 4 receptor (GFP tagged) in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5710)
  AUTHORS   Muratspahic E, Aslanoglou D, White AM, Draxler C, Kozisek X, Farooq 
            Z, Craik DJ, McCormick PJ, Durek T, Gruber CW
  TITLE     Development of Melanocortin 4 Receptor Agonists by Exploiting 
            Animal-Derived Macrocyclic, Disulfide-Rich Peptide Scaffolds.
  JOURNAL   ACS Pharmacol Transl Sci. 2023 Sep 26;6(10):1373-1381. doi: 
            10.1021/acsptsci.3c00090. eCollection 2023 Oct 13.
  PUBMED    37854631
REFERENCE   2  (bases 1 to 5710)
  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"; doi: 
            "10.1021/acsptsci.3c00090"; journalName: "ACS Pharmacol Transl Sci";
            date: "2023-09-26- 26"; volume: "6"; issue: "10"; pages: "1373-1381"
COMMENT     Sequence Label: pEGFP-hMC4R
FEATURES             Location/Qualifiers
     source          1..5710
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1650..1659
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1656..1658,1659..1661,1662..2375)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1656..1658,1659..1661,1662..2372)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1701..1722)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1962..1981)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2309..2330
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2498..2619
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2535..2554)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2589..2608
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2626..3081)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2763..2784)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2975..2994
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3108..3212
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3214..3571
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3214..3405
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3237..3257)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3422..3557
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3606..4400
                     /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             3606..4400
                     /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(3660..3679)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4270..4289
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4588..4607)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4632..4679
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5008..5596
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5497..5516
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcatgg
      601 tgaactccac ccaccgtggg atgcacactt ctctgcacct ctggaaccgc agcagttaca
      661 gactgcacag caatgccagt gagtcccttg gaaaaggcta ctctgatgga gggtgctacg
      721 agcaactttt tgtctctcct gaggtgtttg tgactctggg tgtcatcagc ttgttggaga
      781 atatcttagt gattgtggca atagccaaga acaagaatct gcattcaccc atgtactttt
      841 tcatctgcag cttggctgtg gctgatatgc tggtgagcgt ttcaaatgga tcagaaacca
      901 ttgtcatcac cctattaaac agtacagata cggatgcaca gagtttcaca gtgaatattg
      961 ataatgtcat tgactcggtg atctgtagct ccttgcttgc atccatttgc agcctgcttt
     1021 caattgcagt ggacaggtac tttactatct tctatgctct ccagtaccat aacattatga
     1081 cagttaagcg ggttgggatc atcataagtt gtatctgggc agcttgcacg gtttcaggca
     1141 ttttgttcat catttactca gatagtagtg ctgtcatcat ctgcctcatc accatgttct
     1201 tcaccatgct ggctctcatg gcttctctct atgtccacat gttcctgatg gccaggcttc
     1261 acattaagag gattgctgtc ctccccggca ctggtgccat ccgccaaggt gccaatatga
     1321 agggagcgat taccttgacc atcctgattg gcgtctttgt tgtctgctgg gccccattct
     1381 tcctccactt aatattctac atctcttgtc ctcagaatcc atattgtgtg tgcttcatgt
     1441 ctcactttaa cttgtatctc atactgatca tgtgtaattc aatcatcgat cctctgattt
     1501 atgcactccg gagtcaagaa ctgaggaaaa ccttcaaaga gatcatctgt tgctatcccc
     1561 tgggaggcct ttgtgacttg tctagcagat atgagctcaa gcttcgaatt ctgcagtcga
     1621 cggtaccgcg ggcccgggat ccaccggtcg ccaccatggt gagcaagggc gaggagctgt
     1681 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     1741 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     1801 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
     1861 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     1921 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     1981 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     2041 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     2101 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc
     2161 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
     2221 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga
     2281 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     2341 ggatcactct cggcatggac gagctgtaca agtaaagcgg ccgcgactct agatcataat
     2401 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
     2461 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     2521 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     2581 ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta
     2641 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     2701 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     2761 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     2821 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     2881 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     2941 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     3001 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     3061 atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     3121 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     3181 aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa
     3241 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3301 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     3361 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     3421 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     3481 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     3541 aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt
     3601 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     3661 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     3721 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     3781 aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     3841 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     3901 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     3961 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     4021 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     4081 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc
     4141 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     4201 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     4261 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     4321 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     4381 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     4441 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     4501 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     4561 cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac
     4621 ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca
     4681 taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg
     4741 gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg
     4801 cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata
     4861 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     4921 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     4981 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     5041 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     5101 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     5161 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     5221 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     5281 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     5341 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     5401 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     5461 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     5521 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     5581 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     5641 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     5701 cgccatgcat
//
