LOCUS       sequence-123121-        7117 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pPBC-LG6f expresses PiggyBac inverted terminal repeat-flanked 
            Lck-GCaMP6f under the control of the CAG promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7117)
  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 7117)
  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-LG6f
FEATURES             Location/Qualifiers
     source          1..7117
                     /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      2964..2973
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3087..3098,3099..3116,3117..3119,3120..3152,3153..3155,
                     3156..3179,3180..3206,3207..3263,3264..3269,3270..3539,
                     3540..3557,3558..3992,3993..3998,3999..4442)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWN
                     KTGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDG
                     PVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGE
                     ELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTL
                     TYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRI
                     ELKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEEFSLFDKDGDGTITTKELGTVMR
                     SLGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDG
                     NGYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      3186..3195
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     3476..3497
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3603..3624)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3864..3883)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(4624..4643)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4668..4715
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     repeat_region   5063..5097
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5221..5240)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5222..5240)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5327..5344)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5498..6086)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5578..5597)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6257..7048,7049..7117))
                     /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     6880..6899
                     /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 tctgccacca tgggctgtgg ctgcagctca aaccctgaag
     3001 atgactggat ggagaacatt gacgtgtgtg agaactgcca ttatcccccg ccgaagcttc
     3061 gaattgatcc accggatctc gccaccatgc ggggttctca tcatcatcat catcatggta
     3121 tggctagcat gactggtgga cagcaaatgg gtcgggatct gtacgacgat gacgataagg
     3181 atctcgccac catggtcgac tcatcacgtc gtaagtggaa taagacaggt cacgcagtca
     3241 gagctatagg tcggctgagc tcactcgaga acgtctatat caaggccgac aagcagaaga
     3301 acggcatcaa ggcgaacttc aagatccgcc acaacatcga ggacggcggc gtgcagctcg
     3361 cctaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     3421 actacctgag cgtgcagtcc aaactttcga aagaccccaa cgagaagcgc gatcacatgg
     3481 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg
     3541 gcggtaccgg agggagcatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     3601 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggtg
     3661 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     3721 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     3781 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacatcc
     3841 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     3901 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     3961 gcaacatcct ggggcacaag ctggagtaca acctgccgga ccaactgact gaagagcaga
     4021 tcgcagaatt taaagaggaa ttctccctat ttgacaagga cggggatggg acaataacaa
     4081 ccaaggagct ggggacggtg atgcggtctc tggggcagaa ccccacagaa gcagagctgc
     4141 aggacatgat caatgaagta gatgccgacg gtgacggcac aatcgacttc cctgagttcc
     4201 tgacaatgat ggcaagaaaa atgaaataca gggacacgga agaagaaatt agagaagcgt
     4261 tcggtgtgtt tgataaggat ggcaatggct acatcagtgc agcagagctt cgccacgtga
     4321 tgacaaacct tggagagaag ttaacagatg aagaggttga tgaaatgatc agggaagcag
     4381 acatcgatgg ggatggtcag gtaaactacg aagagtttgt acaaatgatg acagcgaagt
     4441 gagcggccgc ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     4501 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     4561 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct
     4621 aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag
     4681 acagaataaa acgcacggtg ttgggtcgtt tgttcggcgc gccatcgaag cagctccagc
     4741 ctacacaatc gctcaagacg tgtaatgctt ttattatata ttagtcacga tatctataac
     4801 aagaaaatat atatataata agttatcacg taagtagaac atgaaataac aatataatta
     4861 tcgtatgagt taaatcttaa aagtcacgta aaagataatc atgcgtcatt ttgactcacg
     4921 cggtcgttat agttcaaaat cagtgacact taccgcattg acaagcacgc ctcacgggag
     4981 ctccaagcgg cgactgagat gtcctaaatg cacagcgacg gattcgcgct atttagaaag
     5041 agagagcaat atttcaagaa tgcatgcgtc aattttacgc agactatctt tctagggtta
     5101 aaaaagattt gcgctttact cgacctaaac tttaaacacg tcatagaatc ttcgtttgac
     5161 aaaaaccaca ttgtggggta ccgagctcga attcatcgat gatatcagat ctgccggtct
     5221 ccctatagtg agtcgtatta atttcgataa gccaggttaa cctgcattaa tgaatcggcc
     5281 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     5341 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     5401 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5461 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     5521 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5581 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5641 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5701 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5761 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5821 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5881 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     5941 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     6001 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6061 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     6121 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     6181 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6241 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6301 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6361 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6421 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6481 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6541 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6601 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6661 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6721 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6781 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6841 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6901 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6961 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7021 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7081 agggaataag ggcgacacgg aaatgttgaa tactcat
//
