LOCUS       sequence-490776-        7633 bp    DNA     circular SYN 27-OCT-2025
DEFINITION  Empty AAV-RAM plasmid w. multiple cloning site (MCS).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7633)
  AUTHORS   Sorensen AT, Cooper YA, Baratta MV, Weng FJ, Zhang Y, Ramamoorthi K,
            Fropf R, LaVerriere E, Xue J, Young A, Schneider C, Gotzsche CR, 
            Hemberg M, Yin JC, Maier SF, Lin Y
  TITLE     A robust activity marking system for exploring active neuronal 
            ensembles.
  JOURNAL   Elife. 2016 Sep 23;5. pii: e13918. doi: 10.7554/eLife.13918.
  PUBMED    27661450
REFERENCE   2  (bases 1 to 7633)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 27, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2016-09-23"; volume: "5. pii: e13918. doi"; pages: " 
            10.7554/eLife.13918"
COMMENT     Sequence Label: pAAV-RAM-d2TTA::TRE-MCS-WPRE-pA
FEATURES             Location/Qualifiers
     source          1..7633
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      2..456
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(89..108)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     298..319
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        482..586
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(587..655,656..1447)
                     /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(805..824)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1618..2206
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2107..2126
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     mobile_element  2511..2735
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(2802..3335)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3505..3510,3511..3527,3528..3533))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     repeat_region   3873..4002
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        4022..4336
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    4030..4048
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4066..4084
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4101..4119
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4137..4155
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4173..4191
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4208..4226
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4244..4262
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     4309..4333
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     misc_feature    4403..4991
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4456..4476)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4874..4885)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5024..5041)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5030..5141
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    5289..5337
                     /note="synthetic polyadenylation signal"
     misc_feature    5351..5442
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     5391..5410
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     CDS             join(5808..6425,6426..6431,6432..6467,6468..6470,6471..6506,
                     6507..6509,6510..6545,6546..6554)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=tTA-Advanced
                     /note="In the Tet-Off(R) system, tTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     primer_bind     complement(6567..6583)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6608..6627)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6609..6627)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    6653..6787
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   6816..6956
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6816..6945
                     /label=AAV2 ITR
     polyA_signal    7123..7257
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     mobile_element  7286..7451
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
ORIGIN
        1 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
       61 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
      121 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
      181 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
      241 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt cttaatagtg
      301 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
      361 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
      421 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
      481 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
      541 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
      601 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
      661 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
      721 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
      781 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
      841 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
      901 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
      961 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     1021 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     1081 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     1141 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     1201 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     1261 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     1321 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     1381 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     1441 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     1501 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     1561 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     1621 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     1681 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     1741 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     1801 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     1861 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     1921 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     1981 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     2041 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     2101 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     2161 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     2221 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     2281 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     2341 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     2401 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atagaccagc cgcgtaacct
     2461 ggcaaaatcg gttacggttg agtaataaat ggatgccctg cgtaagcggg tgtgggcgga
     2521 caataaagtc ttaaactgaa caaaatagat ctaaactatg acaataaagt cttaaactag
     2581 acagaatagt tgtaaactga aatcagtcca gttatgctgt gaaaaagcat actggacttt
     2641 tgttatggct aaagcaaact cttcattttc tgaagtgcaa attgcccgtc gtattaaaga
     2701 ggggcgtggc caagggcatg gtaaagacta tattcgcggc gttgtgacaa tttaccgaac
     2761 aactccgcgg ccgggaagcc gatctcggct tgaacgaatt gttaggtggc ggtacttggg
     2821 tcgatatcaa agtgcatcac ttcttcccgt atgcccaact ttgtatagag agccactgcg
     2881 ggatcgtcac cgtaatctgc ttgcacgtag atcacataag caccaagcgc gttggcctca
     2941 tgcttgagga gattgatgag cgcggtggca atgccctgcc tccggtgctc gccggagact
     3001 gcgagatcat agatatagat ctcactacgc ggctgctcaa acttgggcag aacgtaagcc
     3061 gcgagagcgc caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta
     3121 cggagcaagt tcccgaggta atcggagtcc ggctgatgtt gggagtaggt ggctacgtct
     3181 ccgaactcac gaccgaaaag atcaagagca gcccgcatgg atttgacttg gtcagggccg
     3241 agcctacatg tgcgaatgat gcccatactt gagccaccta actttgtttt agggcgactg
     3301 ccctgctgcg taacatcgtt gctgctccat aacatcaaac atcgacccac ggcgtaacgc
     3361 gcttgctgct tggatgcccg aggcatagac tgtacaaaaa aacagtcata acaagccatg
     3421 aaaaccgcca ctgcgccgtt accaccgctg cgttcggtca aggttctgga ccagttgcgt
     3481 gagcgcatac gctacttgca ttacagttta cgaaccgaac aggcttatgt caactgggtt
     3541 cgtgccttca tccgtttcca cggtgtgcgt cacccggcaa ccttgggcag cagcgaagtc
     3601 gaggcatttc tgtcctggct ggcgaacgag cgcaaggttt cggtctccac gcatcgtcag
     3661 gcattggcgg ccttgctgtt cttctacggc aaggtgctgt gcacggatct gccctggctt
     3721 caggagatcg gaagacctcg gccgtcgcgg cgcttgccgg tggtgctgac cccggatgaa
     3781 gtggttcgca tcctcggttt tctggaaggc gagcatcgtt tgttcgccca ggactctagc
     3841 tatagttcta gtggttggct acagcttgca tgcctgcagg cagctgcgcg ctcgctcgct
     3901 cactgaggcc gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag
     3961 cgcgcagaga gggagtggcc aactccatca ctaggggttc ctgcggccgc acgcgcgtct
     4021 cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct atcagtgata
     4081 gagaacgatg tcgagtttac tccctatcag tgatagagaa cgtatgtcga gtttactccc
     4141 tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag aacgtatgtc
     4201 gagtttatcc ctatcagtga tagagaacgt atgtcgagtt tactccctat cagtgataga
     4261 gaacgtatgt cgaggtaggc gtgtacggtg ggaggcctat ataagcagag ctcgtttagt
     4321 gaaccgtcag atcgccaccg gtgtttaaac gctagcggcg cgccagcgat ccaccggatc
     4381 tagataactg atccttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac
     4441 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     4501 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     4561 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
     4621 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
     4681 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
     4741 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc
     4801 atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgccctt
     4861 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
     4921 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
     4981 cgcctccccg catcgatacc gtcgactaga gctcgctgat cagcctcgac tgtgccttct
     5041 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     5101 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     5161 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     5221 agcagactag tacgtctctc aaggataagt aagtaatatt aaggtacggg aggtattgga
     5281 caggccgcaa taaaatatct ttattttcat tacatctgtg tgttggtttt ttgtgtgaat
     5341 cgatagtact aacatacgct ctccatcaaa acaaaacgaa acaaaacaaa ctagcaaaat
     5401 aggctgtccc cagtgcaagt gcaggtgcca gaacatttct ctggcctaac tggccgacgc
     5461 gtaccctaga agtttgttcg tgactcagac tagaagtttg ttcgtgactc agactagaag
     5521 tttgttcgtg actcagacta gaagtttgtt cgtgactcag aactcattca taaaacgctt
     5581 gttataaaag cagtggctgc ggcgcctcgt actccaaccg catctgcagc gagcaactga
     5641 gaagccaaga ctgagccggc ggccggatcc agctcatgag ccatggcttc ccgccggagg
     5701 tggaggagca ggatgatggc acgctgccca tgtcttgtgc ccaggagagc gggatggacc
     5761 gtcaccctgc agcctgtgct tctgctagga tcaatgtgga attcaccatg tctagactgg
     5821 acaagagcaa agtcataaac tctgctctgg aattactcaa tgaagtcggt atcgaaggcc
     5881 tgacgacaag gaaactcgct caaaagctgg gagttgagca gcctaccctg tactggcacg
     5941 tgaagaacaa gcgggccctg ctcgatgccc tggcaatcga gatgctggac aggcatcata
     6001 cccacttctg ccccctggaa ggcgagtcat ggcaagactt tctgcggaac aacgccaagt
     6061 cattccgctg tgctctcctc tcacatcgcg acggggctaa agtgcatctc ggcacccgcc
     6121 caacagagaa acagtacgaa accctggaaa atcagctcgc gttcctgtgt cagcaaggct
     6181 tctccctgga gaacgcactg tacgctctgt ccgccgtggg ccactttaca ctgggctgcg
     6241 tattggagga tcaggagcat caagtagcaa aagaggaaag agagacacct accaccgatt
     6301 ctatgccccc acttctgaga caagcaattg agctgttcga ccatcaggga gccgaacctg
     6361 ccttcctttt cggcctggaa ctaatcatat gtggcctgga gaaacagcta aagtgcgaaa
     6421 gcggcgggcc ggccgacgcc cttgacgatt ttgacttaga catgctccca gccgatgccc
     6481 ttgacgactt tgaccttgat atgctgcctg ctgacgctct tgacgatttt gaccttgaca
     6541 tgctccccgg gtaactaagt aagcatcgat accgtcgacc tcgagggggg gcccggtacc
     6601 caattcgccc tatagtgagt cgtattacgc gcgcagcggc cgaccatggc ccaacttgtt
     6661 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     6721 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     6781 ctggatctcc ggacacgtgc ggaccgagcg gccgcaggaa cccctagtga tggagttggc
     6841 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     6901 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggtctg
     6961 agacaataac cctgataaat gcttcaataa tgtaagcttg tcgagaagta ctagaggatc
     7021 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     7081 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     7141 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     7201 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatctga
     7261 tcactgatat cgcctaggag atccgaacca gataagtgaa atctagttcc aaactatttt
     7321 gtcattttta attttcgtat tagcttacga cgctacaccc agttcccatc tattttgtca
     7381 ctcttcccta aataatcctt aaaaactcca tttccacccc tcccagttcc caactatttt
     7441 gtccgcccac agcggggcat ttttcttcct gttatgtttt taatcaaaca tcctgccaac
     7501 tccatgtgac aaaccgtcat cttcggctac tttttctctg tcacagaatg aaaatttttc
     7561 tgtcatctct tcgttattaa tgtttgtaat tgactgaata tcaacgctta tttgcagcct
     7621 gaatggcgaa tgg
//
