LOCUS       Exported                6016 bp ds-DNA     circular SYN 11-MAR-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 6016)
  AUTHORS   Paul Sabatini
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Jan 28, 2021 from SnapGene Viewer 5.2.4
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6016
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     protein_bind    298..568
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        601..638
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     misc_feature    612..632
                     /label=283
     protein_bind    773..806
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    817..850
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites."
     CDS             complement(917..2269)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     primer_bind     1476..1495
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1735..1756
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1862..1883)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2164..2173
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     regulatory      2266..2269
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     regulatory      2270..2275
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(2319..2352)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    complement(2363..2396)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites."
     misc_feature    2420..3008
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2473..2493)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     misc_feature    complement(2477..2496)
                     /label=Feature 21
     CDS             complement(2891..2902)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3043..3060)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3049..3256
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3279..3419
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3279..3408
                     /label=AAV2 ITR
     rep_origin      3494..3949
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3581..3600)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3791..3812
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3966..3985)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4085..4107
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4145..4163)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4231..4335
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4336..5196
                     /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     complement(4554..4573)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5367..5955
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5856..5875
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgaaacaa ttctgcagga attcggggcc
      181 gcggaggctg gatcggtccc ggtgtcttct atggaggtca aaacagcgtg gatggcgtct
      241 ccaggcgatc tgacggttca ctaaacgagc tctgcttata taggtcgagt ttaccactcc
      301 ctatcagtga tagagaaaag tgaaagtcga gtttaccact ccctatcagt gatagagaaa
      361 agtgaaagtc gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac
      421 cactccctat cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata
      481 gagaaaagtg aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga
      541 gtttaccact ccctatcagt gatagagaaa agtgaaagtc gagctcggta cccgggtcga
      601 gtaggcgtgt acggtgggag gcctatataa gcagagctcg tttagtgaac cgtcagatcg
      661 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      721 tccgcggccc cgaattcgag ctcggtaccc ggggatcctc tagtcgacgt cgataacttc
      781 gtatagcata cattatacga agttatccga atcgcaataa cttcgtatag gatactttat
      841 acgaagttat ttggcgcgcc aaatttaaat aacgcaacgt accttcgagg gggcccaagg
      901 aattcttact tgtacgtcac ttcgctgtca tcatttgtac aaactcttcg tagtttacct
      961 gaccatcccc atcgatgtct gcttccctga tcatttcatc aacctcttca tctgttaact
     1021 tctctccaag gtttgtcatc acgtggcgaa gctctgctgc actgatgtag ccattgccat
     1081 ccttatcaaa cacaccgaac gcttctctaa tttcttcttc cgtgtccctg tatttcattt
     1141 ttcttgccat cattgtcagg aactcaggga agtcgattgt gccgtcaccg tcggcatcta
     1201 cttcattgat catgtcctgc agctctgctt ctgtggggtt ctgccccaga gaccgcatca
     1261 ccgtccccag ctccttggtt gttattgtcc catccccgtc cttgtcaaat agggagaaag
     1321 cctctttaaa ttctgcgatc tgctcttcag tcagttggtc cggcaggttg tactccagct
     1381 tgtgccccag gatgttgccg tcctccttga agtcgatgcc cttcagctcg atgcggttca
     1441 ccagggtgtc gccctcgaac ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga
     1501 agaagatggt gcgctcctgg atgtagcctt cgggcatggc ggacttgaag aagtcgtgct
     1561 gcttcatgtg gtcggggtag cggctgaagc actgcacgcc gtaggtcagg gtggtcacga
     1621 gggtgggcca gggcacgggc agcttgccgg tggtgcagat gaacttcagg gtcagcttgc
     1681 cgtaggtggc atcgccctca ccctcgccgg acacgctgaa cttgtggccg tttacgtcgc
     1741 cgtccagctc gaccaggatg ggcaccaccc cggtgaacag ctcctcgccc ttgctcacca
     1801 tgctccctcc ggtaccgccc ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg
     1861 tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt ggggtctttc gaaagtttgg
     1921 actgcacgct caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt
     1981 tctgctggta gtggtaggcg agctgcacgc cgccgtcctc gatgttgtgg cggatgtgga
     2041 agttcgcctt gatgccgttc ttctgcttgt cggccttgat atagacgttc tcgagtgagc
     2101 tcagccgacc tatagctctg actgcgtgac ctgtcttatt ccacttacga cgtgatgagt
     2161 cgaccatggt ggcgagatcc ttatcgtcat cgtcgtacag atcccgaccc atttgctgtc
     2221 caccagtcat gctagccata ccatgatgat gatgatgatg agaacccatg gtggcggatc
     2281 ctaactagct agttggcgcg ccaacctggc cggccttgat aacttcgtat aatgtatgct
     2341 atacgaagtt atccgaatcg caataacttc gtataaagta tcctatacga agttatcgaa
     2401 gatcatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg
     2461 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     2521 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     2581 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     2641 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     2701 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     2761 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     2821 gtcctttcct tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg
     2881 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     2941 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     3001 ctccccgcat cgataccgag cgctgctcga gagatcgatc tgcctcgact gtgccttcta
     3061 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     3121 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     3181 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     3241 gcaggcatgc tggggacacg tgcggaccga gcggccgcag gaacccctag tgatggagtt
     3301 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     3361 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg
     3421 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
     3481 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3541 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3601 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3661 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
     3721 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3781 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcgggctat
     3841 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3901 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     3961 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     4021 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     4081 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     4141 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     4201 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     4261 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     4321 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     4381 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     4441 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     4501 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     4561 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     4621 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     4681 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     4741 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     4801 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     4861 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     4921 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     4981 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     5041 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     5101 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     5161 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     5221 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     5281 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     5341 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     5401 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     5461 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     5521 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     5581 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     5641 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     5701 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     5761 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     5821 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     5881 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     5941 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     6001 ccttttgctc acatgt
//
