LOCUS       Exported                6975 bp ds-DNA     circular SYN 11-AUG-2020
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 6975)
  AUTHORS   Martin Myers
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Jan 28, 2021 from SnapGene Viewer 5.2.4
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6975
                     /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
     primer_bind     62..72
                     /label=ps211
                     /note="vwa5a 5pF1,use w/R2 (214) 493bp"
     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
     primer_bind     612..632
                     /label=ps283
                     /note="cmv minimal"
     primer_bind     763..774
                     /label=ps277
                     /note="WTHN TVA-mcherry-oG F SAlI"
     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."
     primer_bind     853..881
                     /label=BBBB
     primer_bind     853..881
                     /label=PS383
                     /note="mRuby reverse AscI + random Hex"
     CDS             complement(863..1543)
                     /codon_start=1
                     /product="monomeric variant of the red fluorescent protein 
                     eqFP611 (Kredel et al., 2009)"
                     /label=mRuby
                     /translation="MNSLIKENMRMKVVLEGSVNGHQFKCTGEGEGNPYMGTQTMRIKV
                     IEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGGVITV
                     MQDTSLEDGCLVYHAQVRGVNFPSNGAVMQKKTKGWEPNTEMMYPADGGLRGYTHMALK
                     VDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAVAKFA
                     GLGGG"
     primer_bind     1826..1848
                     /label=ps152
                     /note="RosaSynTdT (012570) Syp MUT"
     primer_bind     complement(2326..2348)
                     /label=ps151
                     /note="RosaSynTdT (012570) Syp MUT (523 bp)"
     CDS             complement(2495..2548)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(2549..3265)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     2549..2569
                     /label=ps271
                     /note="WTHN hM3Dq F bsrg1"
     primer_bind     2549..2565
                     /label=PS345
                     /note="FlpO ON/Cre Off WTHN DREADD ChR2 3' NheI"
     primer_bind     complement(2550..2569)
                     /label=PS310
                     /note="3p mCherry out"
     primer_bind     complement(2591..2612)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2815..2834
                     /label=ps180
                     /note="P19 R CRE-dsGFP deepseq"
     primer_bind     2940..2959
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2942..2961
                     /label=ps254
                     /note="hM3Dq R2 (mcitrine)"
     primer_bind     3179..3198
                     /label=ps253
                     /note="hM3Dq R1 (mCitrine)"
     primer_bind     complement(3179..3198)
                     /label=ps177
                     /note="P18 F CRE-dsGFP deepseq,964"
     primer_bind     3180..3199
                     /label=PS373
                     /note="Sun1GFP"
     primer_bind     3199..3220
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3246..3265)
                     /label=PS349
                     /note="FlpO ON/Cre Off WTHN GFP-tettox 5' BsiWi"
     primer_bind     complement(3248..3275)
                     /label=AAAA
                     /note="FseI"
     primer_bind     complement(3248..3275)
                     /label=PS384
                     /note="GFP For FseI + random Hex"
     primer_bind     complement(3248..3267)
                     /label=ps121
                     /note="dsGFP F AscI"
     primer_bind     complement(3248..3266)
                     /label=ps280
                     /note="WTHN eGFP-L10 R nheI"
     primer_bind     complement(3248..3265)
                     /label=ps119
                     /note="dsGFP F AgeI"
     primer_bind     complement(3248..3265)
                     /label=ps117
                     /note="dsGFP F MluI"
     primer_bind     complement(3254..3267)
                     /label=ps274
                     /note="WTHN TETTOX R nhei"
     protein_bind    complement(3278..3311)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    complement(3322..3355)
                     /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    3379..3967
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3432..3452)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     misc_feature    complement(3436..3455)
                     /label=Feature 21
     primer_bind     complement(3436..3455)
                     /label=ps125
                     /note="Ai65 PCS seq R1"
     primer_bind     complement(3454..3473)
                     /label=ps145
                     /note="Rosa26TdT (007909) Mutant Reverse (WPRE)"
     primer_bind     complement(3454..3473)
                     /label=145
     primer_bind     complement(3472..3495)
                     /label=ps126
                     /note="Ai65 PCS seq R2"
     primer_bind     complement(3546..3565)
                     /label=ps178
                     /note="P18 R CRE-dsGFP deepseq"
     primer_bind     complement(3706..3721)
                     /label=ps124
                     /note="R Ai65 MluI test"
     CDS             complement(3850..3861)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4002..4019)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4008..4215
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4024..4043)
                     /label=ps176
                     /note="P1 R CRE-dsGFP deepseq"
     repeat_region   4238..4378
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4238..4367
                     /label=AAV2 ITR
     primer_bind     complement(4307..4317)
                     /label=ps211
                     /note="vwa5a 5pF1,use w/R2 (214) 493bp"
     rep_origin      4453..4908
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4540..4559)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4750..4771
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4925..4944)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5044..5066
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5104..5122)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5190..5294
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5295..6155
                     /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(5513..5532)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6326..6914
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6815..6834
                     /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 taccctccgc ccaggccggc gaacttggcc acggcgtgct
      901 cccgctgcac cacgaacatc tcgttgtcgg actcttccag cctttccagc cggtggtcca
      961 cggcgtggat gccgggcatc ttgatgttgc ccacggtttt cttggatcgg taggtggtca
     1021 cgaagctgca gctcaggtgc cctccgccgt ccaccttcag ggccatgtgg gtgtagcctc
     1081 tcagtccgcc gtcagcgggg tacatcatct cggtgttggg ctcccagccc ttggttttct
     1141 tctgcatcac ggcgccgttg ctgggaaaat tcacgcccct gacctgggca tggtacacca
     1201 ggcagccatc ttccaggctg gtgtcctgca tcacggtgat cacgccgccg tcctcgtatc
     1261 tggtcactct ctcccaggtg aagccctcgg ggaagctctg cttgaagaaa tcggggatgc
     1321 ccttggggta cttgatgaag gtccggctgc cgtacatgaa ggatgtggcc aggatgtcga
     1381 aggcgaaggg cagaggtccg ccctcgatca ctttgatccg catggtctgg gtgcccatgt
     1441 aggggttgcc ctcgccctcg ccggtgcact tgaactggtg gccgttcacg ctgccttcca
     1501 gcaccacctt catccgcatg ttttctttga tcaggctgtt catcgtacgg gtggcgaccg
     1561 gtggatccga catctgattg gagaaggagg tgggcgcacc ctgttggccg tagccttgct
     1621 gcccatagtc gccctgaggc ccgtagccac cgccaccgcc gctggctggc tgcccgtaat
     1681 cgggttgata accaccctga ggcccgtagg agtcctgggg cccatagcct ccggggccct
     1741 gcccatagcc cgcatcgccg taggcatcgc caggagctgg ttgcttttct ggggcgcctg
     1801 gaggtgcgcg catgaatggg gcggcccagc ctgtctcctt gaacacgaac cataggttgc
     1861 caacccagag caccaggttc aggaagccaa acaccaccga ggtgttgagt cctgaagtca
     1921 cagggtccct cagttccttg catgtgtttc ctgtctggcg gcacataggc atctccttga
     1981 taatgttctc tgggtcagtg gccatcttca catcggacag gcctttggcc caggcggatg
     2041 agctaactag ccacatgaaa gcgaacactg ctgtggccag gaagtccatc attggccctt
     2101 tgttgttctc tcggtacttg ttctgcagga agatgtaggt ggccagggcc cccatggagt
     2161 agaggaaggc aaacacagcc acggtgacaa agaattcagc cgaggaggag tagtcaccaa
     2221 ctaggaagat cttggtagtg ccccctttaa cgcaggaggg tgcatcaaag tacacttggt
     2281 gcagcctgaa tgggtactca aattcgactt cgatgttgag ggcactctcc gtcttgttgg
     2341 cacactccac gctcagccga agctctccgg tgtagctgcc gcacgtagca aaggcgaaga
     2401 tggcaaagac ccactgcagc accttcacga agccaagggg ctccttgacc acccggaact
     2461 gacccccagc caccagctga ttcaccacgt ccatggggcc ggggttctcc tccacgtcgc
     2521 cgcaggtcag caggctgccc ctgccctcct tgtacagctc gtccatgccg agagtgatcc
     2581 cggcggcggt cacgaactcc agcaggacca tgtgatcgcg cttctcgttg gggtctttgc
     2641 tcagggcgga ctgggtgctc aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga
     2701 tgggggtgtt ctgctggtag tggtcggcga gctgcacgct gccgtcctcg atgttgtggc
     2761 ggatcttgaa gttcaccttg atgccgttct tctgcttgtc ggccatgata tagacgttgt
     2821 ggctgttgta gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga
     2881 tgcccttcag ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg
     2941 tcttgtagtt gccgtcgtcc ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca
     3001 tggcggactt gaagaagtcg tgctgcttca tgtggtcggg gtagcggctg aagcactgca
     3061 cgccgtaggt cagggtggtc acgagggtgg gccagggcac gggcagcttg ccggtggtgc
     3121 agatgaactt cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc
     3181 tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag gatgggcacc accccggtga
     3241 acagctcctc gcccttgctc accatgggcc ggccttgata acttcgtata atgtatgcta
     3301 tacgaagtta tccgaatcgc aataacttcg tataaagtat cctatacgaa gttatcgaag
     3361 atcatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     3421 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     3481 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     3541 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     3601 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     3661 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     3721 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg
     3781 tcctttcctt ggctgctcgc ctatgttgcc acctggattc tgcgcgggac gtccttctgc
     3841 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     3901 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     3961 tccccgcatc gataccgagc gctgctcgag agatcgatct gcctcgactg tgccttctag
     4021 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     4081 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     4141 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     4201 caggcatgct ggggacacgt gcggaccgag cggccgcagg aacccctagt gatggagttg
     4261 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     4321 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     4381 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     4441 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4501 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     4561 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     4621 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     4681 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     4741 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcgggctatt
     4801 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     4861 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     4921 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     4981 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     5041 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     5101 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     5161 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5221 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5281 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5341 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5401 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5461 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5521 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5581 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5641 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5701 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5761 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5821 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     5881 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     5941 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     6001 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6061 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6121 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     6181 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6241 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6301 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6361 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6421 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     6481 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6541 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6601 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6661 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6721 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6781 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6841 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     6901 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6961 cttttgctca catgt
//
