LOCUS       sequence-178820-        5651 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  pPBC-G6f expresses PiggyBac inverted terminal repeat-flanked GCaMP6f
            under the control of the CAG promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5651)
  AUTHORS   Gee JM, Gibbons MB, Taheri M, Palumbos S, Morris SC, Smeal RM, Flynn
            KF, Economo MN, Cizek CG, Capecchi MR, Tvrdik P, Wilcox KS, White JA
  TITLE     Imaging activity in astrocytes and neurons with genetically encoded 
            calcium indicators following in utero electroporation.
  JOURNAL   Front Mol Neurosci. 2015 Apr 15;8:10. doi: 10.3389/fnmol.2015.00010.
            eCollection 2015.
  PUBMED    25926768
REFERENCE   2  (bases 1 to 5651)
  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: "Front Mol 
            Neurosci."; date: "2015-04-15"; volume: "8:10. doi"; pages: " 
            10.3389/fnmol.2015.00010. eCollection 2015"
COMMENT     Sequence Label: pPBC-G6f
FEATURES             Location/Qualifiers
     source          1..5651
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     39..61
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     123..142
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      189..198
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(195..203,204..221,222..224,225..257,258..260,261..284,
                     285..311,312..368,369..374,375..644,645..662,663..1097,
                     1098..1103,1104..1547)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEEFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      291..300
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     581..602
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(708..729)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(969..988)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(1729..1748)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1773..1820
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     repeat_region   2168..2202
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2326..2345)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2327..2345)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2432..2449)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2603..3191)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2683..2702)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3362..4153,4154..4222))
                     /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     3985..4004
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4223..4327)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4395..4413
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4451..4473)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4573..4592
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4673..4691
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4673..4690
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    4757..4778
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4793..4798,4799..4816,4817..4823)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4831..4847
                     /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)."
     primer_bind     4836..4858
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4855..4871
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4855..4871
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        4892..4910
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   5093..5155
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 gggggacggc tgccttcggg ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac
       61 cggcggctct agagcctctg ctaaccatgt tcatgccttc ttctttttcc tacagctcct
      121 gggcaacgtg ctggttattg tgctgtctca tcattttggc aaagctagcg ctaccggact
      181 cagatctcgc caccatgggt tctcatcatc atcatcatca tggtatggct agcatgactg
      241 gtggacagca aatgggtcgg gatctgtacg acgatgacga taaggatctc gccaccatgg
      301 tcgactcatc acgtcgtaag tggaataaga caggtcacgc agtcagagct ataggtcggc
      361 tgagctcact cgagaacgtc tatatcaagg ccgacaagca gaagaacggc atcaaggcga
      421 acttcaagat ccgccacaac atcgaggacg gcggcgtgca gctcgcctac cactaccagc
      481 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcgtgc
      541 agtccaaact ttcgaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
      601 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagggcggt accggaggga
      661 gcatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
      721 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggtgagggc gatgccacct
      781 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
      841 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
      901 agcagcacga cttcttcaag tccgccatgc ccgaaggcta catccaggag cgcaccatct
      961 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     1021 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     1081 acaagctgga gtacaacctg ccggaccaac tgactgaaga gcagatcgca gaatttaaag
     1141 aggaattctc cctatttgac aaggacgggg atgggacaat aacaaccaag gagctgggga
     1201 cggtgatgcg gtctctgggg cagaacccca cagaagcaga gctgcaggac atgatcaatg
     1261 aagtagatgc cgacggtgac ggcacaatcg acttccctga gttcctgaca atgatggcaa
     1321 gaaaaatgaa atacagggac acggaagaag aaattagaga agcgttcggt gtgtttgata
     1381 aggatggcaa tggctacatc agtgcagcag agcttcgcca cgtgatgaca aaccttggag
     1441 agaagttaac agatgaagag gttgatgaaa tgatcaggga agcagacatc gatggggatg
     1501 gtcaggtaaa ctacgaagag tttgtacaaa tgatgacagc gaagtgagcg gccgcttcga
     1561 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     1621 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     1681 cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa
     1741 ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca
     1801 cggtgttggg tcgtttgttc ggcgcgccat cgaagcagct ccagcctaca caatcgctca
     1861 agacgtgtaa tgcttttatt atatattagt cacgatatct ataacaagaa aatatatata
     1921 taataagtta tcacgtaagt agaacatgaa ataacaatat aattatcgta tgagttaaat
     1981 cttaaaagtc acgtaaaaga taatcatgcg tcattttgac tcacgcggtc gttatagttc
     2041 aaaatcagtg acacttaccg cattgacaag cacgcctcac gggagctcca agcggcgact
     2101 gagatgtcct aaatgcacag cgacggattc gcgctattta gaaagagaga gcaatatttc
     2161 aagaatgcat gcgtcaattt tacgcagact atctttctag ggttaaaaaa gatttgcgct
     2221 ttactcgacc taaactttaa acacgtcata gaatcttcgt ttgacaaaaa ccacattgtg
     2281 gggtaccgag ctcgaattca tcgatgatat cagatctgcc ggtctcccta tagtgagtcg
     2341 tattaatttc gataagccag gttaacctgc attaatgaat cggccaacgc gcggggagag
     2401 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     2461 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     2521 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     2581 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     2641 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     2701 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     2761 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     2821 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     2881 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     2941 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     3001 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     3061 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     3121 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     3181 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     3241 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     3301 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     3361 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     3421 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     3481 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     3541 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     3601 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     3661 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     3721 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     3781 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     3841 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     3901 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     3961 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     4021 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     4081 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     4141 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     4201 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     4261 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     4321 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     4381 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg
     4441 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     4501 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct
     4561 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg gacatattgt
     4621 cgttagaacg cggctacaat taatacataa ccttatgtat catacacata cgatttaggt
     4681 gacactatag aactcgacct cgaggctggc acgacaggtt tcccgactgg aaagcgggca
     4741 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     4801 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     4861 acagctatga ccatgattac gccaagctcg aaattaaccc tcactaaagg gaacaaaagc
     4921 tggagctcgt ctttgatcaa aacgcaaatc gacgaaaatg tgtcggacaa tatcaagtcg
     4981 atgagcgaaa aactaaaaag gctagaatac gacaatctca cagacagcgt tgagatatac
     5041 ggtattcacg acagcaggct gaataataaa aaaattagaa actattattt aaccctagaa
     5101 agataatcat attgtgacgt acgttaaaga taatcatgcg taaaattgac gcatgtgttt
     5161 tatcgatctg tatatcgagg tttatttatt aatttgaata gatattaagt tttattatat
     5221 ttacacttac atactaataa taaattcaac aaacaattta tttatgttta tttatttatt
     5281 aaaaaaaaac aaaaactcaa aatttcttct ataaagtaac aaaactttta aacattctct
     5341 cttttacaaa aataaactta ttttgtactt taaaaacagt catgttgtat tataaaataa
     5401 gtaattagct taacttatac ataatagaaa caaattatac ttattagaca ttgattattg
     5461 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     5521 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     5581 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     5641 caatgggtgg a
//
