LOCUS       sequence-488999-        7610 bp    DNA     circular SYN 09-OCT-2025
DEFINITION  green biosensor for cAMP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7610)
  AUTHORS   Liu W, Liu C, Ren PG, Chu J, Wang L
  TITLE     An Improved Genetically Encoded Fluorescent cAMP Indicator for 
            Sensitive cAMP Imaging and Fast Drug Screening.
  JOURNAL   Front Pharmacol. 2022 May 12;13:902290. doi: 
            10.3389/fphar.2022.902290. eCollection 2022.
  PUBMED    35694242
REFERENCE   2  (bases 1 to 7610)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 9, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Front 
            Pharmacol."; date: "2022-05-12"; volume: "13:902290. doi"; pages: " 
            10.3389/fphar.2022.902290. eCollection 2022"
COMMENT     Sequence Label: pCAG-G-Flamp2
FEATURES             Location/Qualifiers
     source          1..7610
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        92..395
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        397..672
                     /label=chicken beta-actin promoter
     intron          673..1689
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1613..1635
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1697..1716
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1791..1800
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1806..1823
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(1863..1871,1872..1886)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     2327..2348
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2454..2475)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             3114..3143
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3159..3176
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3199..3216)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3205..3429
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3475..3903
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3562..3581)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3772..3793
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3912..3932)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3917..4246
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4097..4232
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4159..4178
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4313..5107
                     /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(4367..4386)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4977..4996
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5283..5404
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5320..5339)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5374..5393
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5453..5469)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5453..5469)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5466..5488)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5477..5493
                     /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(5501..5507,5508..5525,5526..5531))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5546..5567
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5684..5701)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5855..6443)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5935..5954)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6614..7405,7406..7474))
                     /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     7237..7256
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7475..7579)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatcaatt ctgcaggaat ctagttatta atagtaatca attacggggt
       61 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
      121 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
      181 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
      241 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
      301 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
      361 agtacatcta cgtattagtc atcgctatta ccatggtcga ggtgagcccc acgttctgct
      421 tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt attttttaat
      481 tattttgtgc agcgatgggg gcgggggggg ggggggcgcg cgccaggcgg ggcggggcgg
      541 ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg
      601 ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc
      661 gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc
      721 tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac
      781 ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg
      841 tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg
      901 gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc
      961 ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg
     1021 agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc
     1081 gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa
     1141 ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc
     1201 cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg
     1261 ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc
     1321 cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg
     1381 tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg
     1441 cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa
     1501 atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct
     1561 cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc
     1621 ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt
     1681 ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa
     1741 ttgatttgat accgcgggcc cgggatcagc ttggtaccga gctcggatcc gccaccatgg
     1801 gctcccacca ccaccatcac catggcatgg caagcatgac aggggggcag cagatgggac
     1861 gggacctcta tgatgatgac gacaaagatc ccatgggctt ctaccaggag gtgagaagag
     1921 gggactttgt gaggaactgg cagctagtgg cagctgtgcc tctcttccag aagctgggcc
     1981 cagccgtcct ggtagaaatt gtgagagccc tgagagcccg gaccgttcct gctggggctg
     2041 tcatctgtcg cattggagag ccaggagaca ggatgttttt cgttgtggaa ggcagtgtga
     2101 gtgtggctac caactggggc aacgtctata tcacagccga caagcagaat aacggcatca
     2161 aggcgaactt cgagatccgc cacaacgttg agggtggcgg cgtgcagctc gcctaccact
     2221 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     2281 gcgtgcagtc caaactttcg aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     2341 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag ggcggtaccg
     2401 gagggagcat ggtgcgtaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     2461 agctggacgg cgacgtaaac ggccacaagt tcagcgtgcg tggcgagggc gagggcgatg
     2521 ccaccaatgg caagctgagc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     2581 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcgcacgc taccccgacc
     2641 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacatc caggagcgca
     2701 ccatcgtttt caaggacgac ggcacctaca agacccgcgc cgtggtgaag ttcgagggcg
     2761 acaccctggt gaaccgcatc gagctgaagg gcaccgactt caaggaggac ggcaacatcc
     2821 tggggcacaa gctggagtac aaccgggtga atcctgtgga gctgggccct ggagccttct
     2881 ttggggagat ggccttgatc tctggagaac ccagggtggc cactgtctca gctgccacca
     2941 cggtgagcct gctgtctctg cactccgctg atttccaaat gctgtgcagc tcttccccag
     3001 agatcgcaga aatcttcagg aagacagccc tggagagacg aggagcagct gcctcagcct
     3061 aatgaattct gcagatatcc agcacagtgg cggccgctcg aggtcaccca ttcgaacaaa
     3121 aactcatctc agaagaggat ctgaatatgc ataccggtca tcatcaccat caccattgag
     3181 tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     3241 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     3301 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     3361 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     3421 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
     3481 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     3541 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     3601 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     3661 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     3721 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     3781 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     3841 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     3901 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     3961 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     4021 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     4081 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     4141 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     4201 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
     4261 ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga
     4321 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     4381 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     4441 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
     4501 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     4561 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     4621 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     4681 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     4741 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     4801 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga
     4861 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     4921 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     4981 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     5041 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     5101 cttctgagcg ggactctggg gttcgcgaaa tgaccgacca agcgacgccc aacctgccat
     5161 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     5221 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     5281 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     5341 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     5401 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     5461 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     5521 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     5581 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5641 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     5701 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5761 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5821 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5881 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5941 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     6001 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     6061 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     6121 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     6181 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6241 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     6301 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6361 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6421 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6481 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6541 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6601 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6661 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6721 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6781 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6841 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6901 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     6961 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     7021 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     7081 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     7141 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     7201 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     7261 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     7321 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7381 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     7441 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7501 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7561 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
