LOCUS       sequence-243985-        6144 bp    DNA     circular SYN 14-APR-2025
DEFINITION  mammalian expression of Gamillus, an acid-tolerant monomeric green 
            fluorescent protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6144)
  AUTHORS   Shinoda H, Ma Y, Nakashima R, Sakurai K, Matsuda T, Nagai T
  TITLE     Acid-Tolerant Monomeric GFP from Olindias formosa.
  JOURNAL   Cell Chem Biol. 2018 Mar 15;25(3):330-338.e7. doi: 
            10.1016/j.chembiol.2017.12.005. Epub 2017 Dec 28.
  PUBMED    29290624
REFERENCE   2  (bases 1 to 6144)
  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"; doi: 
            "10.1016/j.chembiol.2017.12.005"; journalName: "Cell Chem Biol"; 
            date: "2018-03-15- 15"; volume: "25"; issue: "3"; pages: 
            "330-338.e7"
COMMENT     Sequence Label: Gamillus/ pcDNA3
FEATURES             Location/Qualifiers
     source          1..6144
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        23..402
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        403..606
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     557..577
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     651..670
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        651..669
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             705..1421
                     /codon_start=1
                     /product="acid-tolerant monomeric green fluorescent protein
                     (Shinoda et al., 2018)"
                     /label=Gamillus
                     /translation="MVSKGEEASGRALFQYPMTSKIELNGEINGKKFKVAGEGFTPSSG
                     RFNMHAYCTTGDLPMSWVVIASPLQYGFHMFAHYPEDITHFFQECFPGSYTLDRTLRME
                     GDGTLTTHHEYSLEDGCVTSKTTLNASGFDPKGATMTKSFVKQLPNEVKITPHGPNGIR
                     LTSTVLYLKEDGTIQIGTQDCIVTPVGGRKVTQPKAHFLHTQIIQKKDPNDTRDHIVQT
                     ELAVAGNLWHGMDELYK"
     promoter        complement(1485..1503)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1486..1503)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1523..1540)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1529..1753
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1799..2227
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1886..1905)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2096..2117
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2236..2256)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2241..2570
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2421..2556
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2483..2502
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2637..3431
                     /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(2691..2710)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3301..3320
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3605..3726
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3642..3661)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3696..3715
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3775..3791)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3775..3791)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3788..3810)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3799..3815
                     /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(3823..3829,3830..3847,3848..3853))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3868..3889
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4006..4023)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4177..4765)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4257..4276)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4936..5727,5728..5796))
                     /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     5559..5578
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5797..5901)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5976..5995)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
       61 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
      121 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
      181 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
      241 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
      301 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
      361 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
      421 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
      481 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
      541 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
      601 agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat taatacgact
      661 cactataggg agacccaagc ttggtaccga gctcggatcc caccatggtg agcaagggcg
      721 aggaggcatc tggcagagcc ctgttccagt accccatgac cagcaagatc gagctgaacg
      781 gcgagatcaa cggcaagaaa ttcaaggtgg ccggcgaggg cttcaccccc agcagcggca
      841 gattcaacat gcacgcctac tgcaccaccg gcgacctgcc tatgagctgg gtcgtgattg
      901 ccagccccct ccagtacggc ttccacatgt tcgcccacta ccccgaggac atcacacact
      961 ttttccagga atgcttcccc ggcagctaca ccctggaccg gaccctgaga atggaaggcg
     1021 acggcaccct gaccacccac cacgagtaca gcctggagga cggctgcgtg acctccaaga
     1081 ccaccctgaa tgccagcggc ttcgacccta agggcgccac catgaccaag agcttcgtga
     1141 aacaactgcc taacgaggtg aagatcaccc cccacggccc caacggcatc agactgacca
     1201 gcaccgtgct gtacctgaag gaggatggca ccatccagat cggcacccag gactgcatcg
     1261 tgacccctgt gggcggaagg aaagtgaccc agcccaaggc ccacttcctg cacacccaga
     1321 tcatccagaa gaaggacccc aacgacaccc gggaccacat cgtgcagaca gaactggccg
     1381 tggccggcaa tctgtggcac ggcatggacg agctgtacaa gtaagaattc tgcagatatc
     1441 catcacactg gcggccgctc gagcatgcat ctagagggcc ctattctata gtgtcaccta
     1501 aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     1561 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     1621 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     1681 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     1741 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac
     1801 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     1861 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     1921 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     1981 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     2041 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     2101 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     2161 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     2221 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     2281 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     2341 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     2401 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     2461 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
     2521 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
     2581 gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga
     2641 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     2701 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     2761 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg
     2821 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     2881 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     2941 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     3001 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     3061 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     3121 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg
     3181 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     3241 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     3301 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     3361 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     3421 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     3481 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     3541 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     3601 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     3661 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     3721 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     3781 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     3841 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     3901 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     3961 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     4021 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4081 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4141 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4201 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4261 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4321 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4381 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4441 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4501 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4561 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     4621 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4681 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4741 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4801 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     4861 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     4921 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     4981 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     5041 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     5101 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     5161 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     5221 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     5281 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     5341 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     5401 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     5461 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     5521 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     5581 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     5641 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     5701 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     5761 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     5821 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     5881 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc
     5941 gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag
     6001 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa
     6061 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt
     6121 aggcgttttg cgctgcttcg cgat
//
