LOCUS       sequence-60327-e        5941 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5941)
  AUTHORS   Shigetomi E, Bushong EA, Haustein MD, Tong X, Jackson-Weaver O, 
            Kracun S, Xu J, Sofroniew MV, Ellisman MH, Khakh BS
  TITLE     Imaging calcium microdomains within entire astrocyte territories and
            endfeet with GCaMPs expressed using adeno-associated viruses.
  JOURNAL   J Gen Physiol. 2013 Apr 15.
  PUBMED    23589582
REFERENCE   2  (bases 1 to 5941)
  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: "J Gen 
            Physiol. 2013 Apr 15."
COMMENT     Sequence Label: pZac2.1 gfaABC1D-Lck-GCaMP3
FEATURES             Location/Qualifiers
     source          1..5941
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   259..388
                     /label=AAV2 ITR
     intron          1239..1371
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1354..1373
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1416..1434
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(1571..1582,1583..1600,1601..1603,1604..1636,1637..1639,
                     1640..1663,1664..1690,1691..1747,1748..1753,1754..2023,
                     2024..2041,2042..2476,2477..2479,2480..2482,2483..2926)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWN
                     KTGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDG
                     PVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGE
                     ELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTL
                     TYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRI
                     ELKGIDFKEDGNILGHKLEYNTRDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMR
                     SLGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDG
                     NGYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     CDS             join(1571..1582,1583..1600,1601..1603,1604..1636,1637..1639,
                     1640..1663,1664..1690,1691..1747,1748..1753,1754..2023,
                     2024..2041,2042..2476,2477..2479,2480..2482,2483..2926)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWN
                     KTGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDG
                     PVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGE
                     ELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTL
                     TYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRI
                     ELKGIDFKEDGNILGHKLEYNTRDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMR
                     SLGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDG
                     NGYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      1670..1679
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1960..1981
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2087..2108)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2348..2367)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     polyA_signal    2966..3087
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2977..2996)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3031..3050
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3249..3378
                     /label=AAV2 ITR
     primer_bind     complement(3397..3414)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3397..3413)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3406..3428)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3555..4010
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3642..3661)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3852..3873
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4036..4140
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4141..4209,4210..5001)
                     /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"
     rep_origin      5172..5760
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5661..5680
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccagattta
      241 attaaggcct taattaggct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc
      301 gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga
      361 gtggccaact ccatcactag gggttccttg tagttaatga ttaacccgcc atgctactta
      421 tctacgtagc catgctctag gaagatctaa catatcctgg tgtggagtag gggacgctgc
      481 tctgacagag gctcgggggc ctgagctggc tctgtgagct ggggaggagg cagacagcca
      541 ggccttgtct gcaagcagac ctggcagcat tgggctggcc gccccccagg gcctcctctt
      601 catgcccagt gaatgactca ccttggcaca gacacaatgt tcggggtggg cacagtgcct
      661 gcttcccgcc gcaccccagc ccccctcaaa tgccttccga gaagcccatt gagcaggggg
      721 cttgcattgc accccagcct gacagcctgg catcttggga taaaagcagc acagccccct
      781 aggggctgcc cttgctgtgt ggcgccaccg gcggtggaga acaaggctct attcagcctg
      841 tgcccaggaa aggggatcag gggatgccca ggcatggaca gtgggtggca gggggggaga
      901 ggagggctgt ctgcttccca gaagtccaag gacacaaatg ggtgagggga gagctctccc
      961 catagctggg ctgcggccca accccacccc ctcaggctat gccagggggt gttgccaggg
     1021 gcacccgggc atcgccagtc tagcccactc cttcataaag ccctcgcatc ccaggagcga
     1081 gcagagccag agcaggttgg agaggagacg catcacctcc gctgctcgca agctttattg
     1141 cggtagttta tcacagttaa attgctaacg cagtcagtgc ttctgacaca acagtctcga
     1201 acttaagctg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag gttacaagac
     1261 aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct
     1321 gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca ggtgtccact
     1381 cccagttcaa ttacagctct taaggctaga gtacttaata cgactcacta taggctagcc
     1441 tcgagaattc accatgggct gtggctgcag ctcaaaccct gaagatgact ggatggagaa
     1501 cattgacgtg tgtgagaact gccattatcc cccgccgaag cttcgaattg atccaccgga
     1561 tctcgccacc atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg
     1621 tggacagcaa atgggtcggg atctgtacga cgatgacgat aaggatctcg ccaccatggt
     1681 cgactcatca cgtcgtaagt ggaataagac aggtcacgca gtcagagcta taggtcggct
     1741 cagctcactc gagaacgtct atatcaaggc cgacaagcag aagaacggca tcaaggcgaa
     1801 cttcaagatc cgccacaaca tcgaggacgg cggcgtgcag ctcgcctacc actaccagca
     1861 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcgtcca
     1921 gtccaaactt tcgaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     1981 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagggcggaa ccggagggag
     2041 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     2101 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggtgagggcg atgccaccta
     2161 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     2221 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     2281 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac atccaggagc gcaccatctt
     2341 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     2401 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     2461 caagctggag tacaacacgc gtgaccaact gactgaagag cagatcgcag aatttaaaga
     2521 ggctttctcc ctatttgaca aggacgggga tgggacaata acaaccaagg agctggggac
     2581 ggtgatgcgg tctctggggc agaaccccac agaagcagag ctgcaggaca tgatcaatga
     2641 agtagatgcc gacggtgatg gcacaatcga cttccctgag ttcctgacaa tgatggcaag
     2701 aaaaatgaaa gacacagaca gtgaagaaga aattagagaa gcgttccgtg tgtttgataa
     2761 ggatggcaat ggctacatca gtgcagcaga gcttcgccac gtgatgacaa accttggaga
     2821 gaagttaaca gatgaagagg ttgatgaaat gatcagggaa gcagacatcg atggggatgg
     2881 tcaggtaaac tacgaagagt ttgtacaaat gatgacagcg aagtaatcta gagtcgaccc
     2941 gggcggccgc ttcgagcaga catgataaga tacattgatg agtttggaca aaccacaact
     3001 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     3061 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     3121 gttcaggggg agatgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtaaa
     3181 atcgataagg atcttcctag agcatggcta cgtagataag tagcatggcg ggttaatcat
     3241 taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     3301 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     3361 gagcgagcga gcgcgcagcc ttaattaacc taattcactg gccgtcgttt tacaacgtcg
     3421 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     3481 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     3541 gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3601 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     3661 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3721 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3781 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3841 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     3901 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3961 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     4021 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     4081 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     4141 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     4201 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     4261 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     4321 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     4381 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     4441 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     4501 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     4561 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     4621 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     4681 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     4741 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     4801 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     4861 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     4921 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     4981 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     5041 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     5101 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     5161 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     5221 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     5281 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     5341 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     5401 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     5461 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     5521 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     5581 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     5641 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     5701 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     5761 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     5821 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     5881 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     5941 g
//
