LOCUS       Syn_AAV_RPTEN_CD        6933 bp DNA     circular SYN 14-FEB-2023
DEFINITION  Cut Synapsin vector with BamHI and HindIII.
            PCR G/RPTEN Insert with custom primers (Syn_PTEN_IFor/IRev).
            Gibson.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV.Syn.GCaMP6f.WPRE.SV40
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6933)
  AUTHORS   Chen TW, Wardill TJ, Sun Y, Pulver SR, Renninger SL, Baohan A, 
            Schreiter ER, Kerr RA, Orger MB, Jayaraman V, Looger LL, Svoboda K, 
            Kim DS
  TITLE     Ultrasensitive fluorescent proteins for imaging neuronal activity.
  JOURNAL   Nature. 2013 Jul 18;499(7458):295-300. doi: 10.1038/nature12354.
  PUBMED    23868258
REFERENCE   2  (bases 1 to 6933)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 24, 2024 from SnapGene 7.0.2
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10"; journalName: 
            "Nature"; date: "2013-07-18- 18"; volume: "499"; issue: "7458"; 
            pages: "295-300"
FEATURES             Location/Qualifiers
     source          1..6933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     229..268
                     /label=Syn_PTEN_IFor
                     /note="color: black; sequence: 
                     gaattcaagctgctagcaagccaccATGGTGAGCAAGGGC; added: 
                     2023-02-07"
     misc_feature    254..955
                     /label=mCyRFP2
                     /note="color: #a6acb3"
     misc_feature    977..2185
                     /label=PTEN
                     /note="color: #a6acb3"
     misc_feature    2186..2194
                     /label=linker PTP
                     /note="color: #a6acb3"
     misc_feature    2195..2923
                     /label=CDmMaroon1
                     /note="color: #a6acb3"
     primer_bind     complement(2908..2947)
                     /label=Primer 1
                     /note="color: black; sequence: 
                     gaggttgattatcgataagctttaCTTATACAGTTCATCC; added: 
                     2023-02-14"
     CDS             2924..2926
                     /codon_start=1
                     /product="bright, rapidly maturing, monomeric far-red 
                     fluorescent protein (Bajar et al., 2016)"
                     /label=mMaroon1
                     /note="color: #a0003c"
                     /translation=""
     misc_feature    2938..3526
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
                     /note="color: #ffffff"
     primer_bind     complement(2991..3011)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
                     /note="color: black; sequence: CATAGCGTAAAAGGAGCAACA"
     CDS             complement(3409..3420)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /note="color: #cc99b2
                     Cleavage site after base 3408"
                     /translation="IEGR"
     primer_bind     complement(3529..3545)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     primer_bind     complement(3530..3546)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
                     /note="color: black; sequence: TCGAGGTCGACGGTATC"
     polyA_signal    3575..3696
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
                     /note="color: #a6acb3"
     primer_bind     complement(3586..3605)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
                     /note="color: black; sequence: GTGGTTTGTCCAAACTCATC"
     primer_bind     3640..3659
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
                     /note="color: black; sequence: GAAATTTGTGATGCTATTGC"
     repeat_region   3858..3987
                     /label=AAV2 ITR
                     /note="color: #ffcc99"
     primer_bind     complement(3904..3915)
                     /label=L4440
                     /note="L4440 vector, forward primer"
                     /note="color: black; sequence: AGCGAGTCAGTGAGCGAG"
     primer_bind     3965..3976
                     /label=L4440
                     /note="L4440 vector, forward primer"
                     /note="color: black; sequence: AGCGAGTCAGTGAGCGAG"
     primer_bind     complement(4053..4070)
                     /label=L4440
                     /note="L4440 vector, forward primer"
                     /note="color: black; sequence: AGCGAGTCAGTGAGCGAG"
     rep_origin      complement(4224..4812)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     primer_bind     complement(4304..4323)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
                     /note="color: black; sequence: GGGAAACGCCTGGTATCTTT"
     CDS             complement(4983..5843)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 4983 .. 5774 / #ccffcc
                        2: 5775 .. 5843 / #ccffcc / signal sequence
                     Cleavage site after base 5774"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5606..5625
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
                     /note="color: black; sequence: ATAATACCGCGCCACATAGC"
     promoter        complement(5844..5948)
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: LEFT"
     rep_origin      complement(5974..6429)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     primer_bind     complement(6111..6132)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
                     /note="color: black; sequence: GTGGACTCTTGTTCCAAACTGG"
     primer_bind     6323..6342
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
                     /note="color: black; sequence: AGGGAAGAAAGCGAAAGGAG"
     repeat_region   6501..6630
                     /label=AAV2 ITR
                     /note="color: #ffcc99"
     primer_bind     complement(6512..6523)
                     /label=L4440
                     /note="L4440 vector, forward primer"
                     /note="color: black; sequence: AGCGAGTCAGTGAGCGAG"
     primer_bind     6573..6584
                     /label=L4440
                     /note="L4440 vector, forward primer"
                     /note="color: black; sequence: AGCGAGTCAGTGAGCGAG"
     promoter        join(6712..6933,1..226)
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
                     /note="color: #ffffff; direction: RIGHT"
ORIGIN
        1 cctcagcact gaaggcgcgc tgacgtcact cgccggtccc ccgcaaactc cccttcccgg
       61 ccaccttggt cgcgtccgcg ccgccgccgg cccagccgga ccgcaccacg cgaggcgcga
      121 gatagggggg cacgggcgcg accatctgcg ctgcggcgcc ggcgactcag cgctgcctca
      181 gtctgcggtg ggcagcggag gagtcgtgtc gtgcctgaga gcgcagtcga attcaagctg
      241 ctagcaagcc accatggtga gcaagggcga ggagctgatc aaggaggaaa tgaccagcaa
      301 gctgtacctg gaaggcagcg tgaacggcca ctacttcaag tgcacccacg aaggggaggg
      361 caagccctac gagggcaagc agaccgccag gatcaaggtg gtggagggag gccccctgcc
      421 gttcgcattc gacatcctgg ccacccactt tatgtacggg agcaaggtgt tcatcaagta
      481 ccccgccgac atccccgatt attttaagca gtccttccct gagggcttca catgggagag
      541 agtcatggtg ttcgaagacg ggggcgtgct gaccactacc caggacacca gcctccagga
      601 cggcgtgctc atctacaacg tcaagaccag aggggtgaac ttcccagcca acggccccgt
      661 gatgcagaag aaaacactgg gctgggagcc cagcaccgag accatgtacc ccgccgacgg
      721 cggcctggaa ggcagatgcg acaagaagct gaagctcgtg ggcgggggcc acctgcacgt
      781 caacttcaag accacataca agtccaagaa acccgtgaag atgcccggcg tccactacgt
      841 ggacaggaga ctggaaagaa tcaaggaggc cgacaacgag acctacgtcg agcagtacga
      901 gcacgctgtg gccagatact ccaacctggg cggaggcatg gacgagctgt acaagtccgg
      961 actcagatct cgagctatga cagccatcat caaagagatc gttagcagaa acaaaaggag
     1021 atatcaagag gatggattcg acttagactt gacctatatt tatccaaata ttattgctat
     1081 gggatttcct gcagaaagac ttgaaggtgt atacaggaac aatattgatg atgtagtaag
     1141 gtttttggat tcaaagcata aaaaccatta caagatatac aatctatgtg ctgagagaca
     1201 ttatgacacc gccaaattta actgcagagt tgcacagtat ccttttgaag accataaccc
     1261 accacagcta gaacttatca aacccttttg tgaagatctt gaccaatggc taagtgaaga
     1321 cgacaatcat gttgcagcaa ttcactgtaa agctgggaaa ggacggactg gtgtaatgat
     1381 ttgtgcatat ttattgcatc ggggcaagtt tttaaaggca caagaggccc tggattttta
     1441 tggggaagta aggaccagag ataaaaaggg agtaactatt cccagtcaga ggcgctatgt
     1501 atattattat agctacctgt taaagaatca cctggattac agaccagtgg cactgttgtt
     1561 tcacaagatg atgtttgaaa ctattccaat gttcagtggc ggaacttgca atccccagtt
     1621 tgtggtctgc cagctaaagg tgaagatcta ctcctccaac tcaggaccca cgcggcggga
     1681 ggacaagctc atgtactttg agttccctca gccattgcct gtgtgtggtg acatcaaagt
     1741 agagttcttc cacaaacaga acaagatgct caaaaaggac aaaatgtttc acttttgggt
     1801 aaatacgttc ttcataccag gaccagagga aacctcagaa aaagtggaaa atggaagtct
     1861 ttgtgatcag gaaatcgata gcatttgtag tatagagcgt gcggataatg acaaggagta
     1921 tcttgtgctc accctgacaa aaaatgatct tgacaaagca aacaaagaca aggccaaccg
     1981 atacttctct ccaaatttta aggtgaaatt atactttaca aaaacagtag aggaaccatc
     2041 aaatccagag gctagcagtt caacttctgt gactccagac gttagtgaca atgaacctga
     2101 tcattataga tattctgaca ccactgactc tgatccagag aatgaacctt ttgatgaaga
     2161 tcagcattca caaattacaa aagtccctac ccctgtcagt aagggtgagg aactgattaa
     2221 agaaaatatg catacaaaac tctatctgac cggaacagtt aataatcatt actttgaatg
     2281 tactgctgaa ggtgaaggaa aaccttatga gggtactcaa actaatagaa ttaaagtcgt
     2341 taggggtggt ccactccctt ttgcttttga tattctcgct ccatgtttta tgtatggttc
     2401 aaaaacattt attaatcatc ctcctgatat accagattat ttcaaacaat cttttccaga
     2461 aggattcact tgggaaagaa ctactgttta tgaagatggg ggaactctca cagcaacaca
     2521 agatacatca ctccaagatg gatgtctgat ttataatgtt caagttagag gtgagaattt
     2581 tcctagtaac ggacctgtta tgcaaaaaaa gacactgggt tgggaagcta gtacagagac
     2641 tctctatcca gcagatggta gtctcgaagg taggctcgat tgggctctga aactggttgg
     2701 tggtggacat ctccactgtc gcctcgaaac tacatataga agtaaaaagc cagctaaaaa
     2761 tctcaaaatg ccaggtgttt atttcataga tagaagactc gaaaggataa aagaagcaga
     2821 taatgaaaca tacgtggaac aacatgaagt tgcagttgct agatattgtg atctgccaag
     2881 taaactcgga cataaactga atggtatgga tgaactgtat aagtaaagct tatcgataat
     2941 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     3001 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     3061 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     3121 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     3181 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     3241 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     3301 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     3361 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     3421 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     3481 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgtcga
     3541 cctcgacccg ggcggccgct tcgagcagac atgataagat acattgatga gtttggacaa
     3601 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
     3661 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt
     3721 atgtttcagg ttcaggggga gatgtgggag gttttttaaa gcaagtaaaa cctctacaaa
     3781 tgtggtaaaa tcgataagga tcttcctaga gcatggctac gtagataagt agcatggcgg
     3841 gttaatcatt aactacaagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     3901 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     3961 ggcctcagtg agcgagcgag cgcgcagctg cattaatgaa tcggccaacg cgcggggaga
     4021 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4081 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4141 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4201 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4261 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4321 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4381 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4441 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4501 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4561 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4621 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     4681 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4741 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4801 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     4861 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     4921 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     4981 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5041 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5101 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5161 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5221 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5281 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5341 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     5401 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     5461 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     5521 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     5581 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     5641 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     5701 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     5761 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     5821 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     5881 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     5941 gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa
     6001 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     6061 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     6121 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     6181 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     6241 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     6301 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     6361 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     6421 agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     6481 cctcttcgct attacgccag ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc
     6541 ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg
     6601 gagtggccaa ctccatcact aggggttcct tgtagttaat gattaacccg ccatgctact
     6661 tatctacgta gccatgctct aggaagatct ctgcagaggg ccctgcgtat gagtgcaagt
     6721 gggttttagg accaggatga ggcggggtgg gggtgcctac ctgacgaccg accccgaccc
     6781 actggacaag cacccaaccc ccattcccca aattgcgcat cccctatcag agagggggag
     6841 gggaaacagg atgcggcgag gcgcgtgcgc actgccagct tcagcaccgc ggacagtgcc
     6901 ttcgcccccg cctggcggcg cgcgccaccg ccg
//
