LOCUS       sequence-123116-        6998 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pPBC-G6f expresses PiggyBac inverted terminal repeat-flanked GCaMP6f
            under the control of the CAG promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6998)
  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 6998)
  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..6998
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     173..191
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(229..251)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     351..370
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        451..469
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     451..468
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    535..556
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(571..576,577..594,595..601)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    609..625
                     /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     614..636
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     633..649
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     633..649
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        670..688
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   871..933
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        1225..1604
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1607..1882
                     /label=chicken beta-actin promoter
     intron          1883..2891
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2815..2837
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2899..2918
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2965..2974
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2971..2979,2980..2997,2998..3000,3001..3033,3034..3036,
                     3037..3060,3061..3087,3088..3144,3145..3150,3151..3420,
                     3421..3438,3439..3873,3874..3879,3880..4323)
                     /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      3067..3076
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     3357..3378
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3484..3505)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3745..3764)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(4505..4524)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4549..4596
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     repeat_region   4944..4978
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5102..5121)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5103..5121)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5208..5225)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5379..5967)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5459..5478)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6138..6929,6930..6998))
                     /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     6761..6780
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
      181 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
      241 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
      301 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
      361 gcagattgta ctgagagtgc accatatgga catattgtcg ttagaacgcg gctacaatta
      421 atacataacc ttatgtatca tacacatacg atttaggtga cactatagaa ctcgacctcg
      481 aggctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
      541 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
      601 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
      661 caagctcgaa attaaccctc actaaaggga acaaaagctg gagctcgtct ttgatcaaaa
      721 cgcaaatcga cgaaaatgtg tcggacaata tcaagtcgat gagcgaaaaa ctaaaaaggc
      781 tagaatacga caatctcaca gacagcgttg agatatacgg tattcacgac agcaggctga
      841 ataataaaaa aattagaaac tattatttaa ccctagaaag ataatcatat tgtgacgtac
      901 gttaaagata atcatgcgta aaattgacgc atgtgtttta tcgatctgta tatcgaggtt
      961 tatttattaa tttgaataga tattaagttt tattatattt acacttacat actaataata
     1021 aattcaacaa acaatttatt tatgtttatt tatttattaa aaaaaaacaa aaactcaaaa
     1081 tttcttctat aaagtaacaa aacttttaaa cattctctct tttacaaaaa taaacttatt
     1141 ttgtacttta aaaacagtca tgttgtatta taaaataagt aattagctta acttatacat
     1201 aatagaaaca aattatactt attagacatt gattattgac tagttattaa tagtaatcaa
     1261 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     1321 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     1381 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggac tatttacggt
     1441 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     1501 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     1561 ctacttggca gtacatctac gtattagtca tcgctattac catgggtcga ggtgagcccc
     1621 acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt
     1681 attttttaat tattttgtgc agcgatgggg gcgggggggg ggggggcgcg cgccaggcgg
     1741 ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca
     1801 gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa
     1861 aaagcgaagc gcgcggcggg cgggagtcgc tgcgttgcct tcgccccgtg ccccgctccg
     1921 cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg
     1981 gcgggacggc ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggctcgtttc
     2041 ttttctgtgg ctgcgtgaaa gccttaaagg gctccgggag ggccctttgt gcggggggga
     2101 gcggctcggg gggtgcgtgc gtgtgtgtgt gcgtggggag cgccgcgtgc ggcccgcgct
     2161 gcccggcggc tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gcgtgtgcgc
     2221 gaggggagcg cggccggggg cggtgccccg cggtgcgggg gggctgcgag gggaacaaag
     2281 gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg gcggtcgggc
     2341 tgtaaccccc ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg
     2401 gggctccgtg cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg
     2461 ggggtgccgg gcggggcggg gccgcctcgg gccggggagg gctcggggga ggggcgcggc
     2521 ggccccggag cgccggcggc tgtcgaggcg cggcgagccg cagccattgc cttttatggt
     2581 aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat ctggcggagc cgaaatctgg
     2641 gaggcgccgc cgcaccccct ctagcgggcg cgggcgaagc ggtgcggcgc cggcaggaag
     2701 gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccatctccag
     2761 cctcggggct gccgcagggg gacggctgcc ttcggggggg acggggcagg gcggggttcg
     2821 gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat gccttcttct
     2881 ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat tttggcaaag
     2941 ctagcgctac cggactcaga tctcgccacc atgggttctc atcatcatca tcatcatggt
     3001 atggctagca tgactggtgg acagcaaatg ggtcgggatc tgtacgacga tgacgataag
     3061 gatctcgcca ccatggtcga ctcatcacgt cgtaagtgga ataagacagg tcacgcagtc
     3121 agagctatag gtcggctgag ctcactcgag aacgtctata tcaaggccga caagcagaag
     3181 aacggcatca aggcgaactt caagatccgc cacaacatcg aggacggcgg cgtgcagctc
     3241 gcctaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     3301 cactacctga gcgtgcagtc caaactttcg aaagacccca acgagaagcg cgatcacatg
     3361 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     3421 ggcggtaccg gagggagcat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     3481 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggt
     3541 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     3601 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     3661 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacatc
     3721 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     3781 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     3841 ggcaacatcc tggggcacaa gctggagtac aacctgccgg accaactgac tgaagagcag
     3901 atcgcagaat ttaaagagga attctcccta tttgacaagg acggggatgg gacaataaca
     3961 accaaggagc tggggacggt gatgcggtct ctggggcaga accccacaga agcagagctg
     4021 caggacatga tcaatgaagt agatgccgac ggtgacggca caatcgactt ccctgagttc
     4081 ctgacaatga tggcaagaaa aatgaaatac agggacacgg aagaagaaat tagagaagcg
     4141 ttcggtgtgt ttgataagga tggcaatggc tacatcagtg cagcagagct tcgccacgtg
     4201 atgacaaacc ttggagagaa gttaacagat gaagaggttg atgaaatgat cagggaagca
     4261 gacatcgatg gggatggtca ggtaaactac gaagagtttg tacaaatgat gacagcgaag
     4321 tgagcggccg cttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     4381 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     4441 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccaccct agggggaggc
     4501 taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa
     4561 gacagaataa aacgcacggt gttgggtcgt ttgttcggcg cgccatcgaa gcagctccag
     4621 cctacacaat cgctcaagac gtgtaatgct tttattatat attagtcacg atatctataa
     4681 caagaaaata tatatataat aagttatcac gtaagtagaa catgaaataa caatataatt
     4741 atcgtatgag ttaaatctta aaagtcacgt aaaagataat catgcgtcat tttgactcac
     4801 gcggtcgtta tagttcaaaa tcagtgacac ttaccgcatt gacaagcacg cctcacggga
     4861 gctccaagcg gcgactgaga tgtcctaaat gcacagcgac ggattcgcgc tatttagaaa
     4921 gagagagcaa tatttcaaga atgcatgcgt caattttacg cagactatct ttctagggtt
     4981 aaaaaagatt tgcgctttac tcgacctaaa ctttaaacac gtcatagaat cttcgtttga
     5041 caaaaaccac attgtggggt accgagctcg aattcatcga tgatatcaga tctgccggtc
     5101 tccctatagt gagtcgtatt aatttcgata agccaggtta acctgcatta atgaatcggc
     5161 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     5221 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     5281 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5341 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     5401 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5461 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5521 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     5581 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5641 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5701 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5761 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     5821 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5881 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5941 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6001 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     6061 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     6121 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     6181 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6241 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     6301 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     6361 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     6421 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     6481 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6541 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6601 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6661 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6721 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6781 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6841 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6901 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6961 aagggaataa gggcgacacg gaaatgttga atactcat
//
