LOCUS       Exported                6266 bp ds-DNA     circular SYN 28-FEB-2020
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 6266)
  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..6266
                     /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."
     CDS             complement(906..911)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="K"
     primer_bind     910..937
                     /label=Primer 2
     primer_bind     complement(1184..1203)
                     /label=PS367
                     /note="caspase 3 screening"
     CDS             complement(1622..1675)
                     /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(1991..2011)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(2514..2544)
                     /label=Primer 1
     regulatory      2530..2534
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(2569..2602)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    complement(2613..2646)
                     /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    2670..3258
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2723..2743)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     misc_feature    complement(2727..2746)
                     /label=Feature 21
     primer_bind     complement(2727..2746)
                     /label=ps125
                     /note="Ai65 PCS seq R1"
     primer_bind     complement(2745..2764)
                     /label=ps145
                     /note="Rosa26TdT (007909) Mutant Reverse (WPRE)"
     primer_bind     complement(2763..2786)
                     /label=ps126
                     /note="Ai65 PCS seq R2"
     primer_bind     complement(2837..2856)
                     /label=ps178
                     /note="P18 R CRE-dsGFP deepseq"
     primer_bind     complement(2997..3012)
                     /label=ps124
                     /note="R Ai65 MluI test"
     CDS             complement(3141..3152)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3293..3310)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3299..3506
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3315..3334)
                     /label=ps176
                     /note="P1 R CRE-dsGFP deepseq"
     repeat_region   3529..3669
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3529..3658
                     /label=AAV2 ITR
     primer_bind     complement(3598..3608)
                     /label=ps211
                     /note="vwa5a 5pF1,use w/R2 (214) 493bp"
     rep_origin      3744..4199
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3831..3850)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4041..4062
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4216..4235)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4335..4357
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4395..4413)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4481..4585
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4586..5446
                     /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(4804..4823)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5617..6205
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6106..6125
                     /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 tgtacatcaa agctgagtgg cttccttaac gggctggaag ggttcctcgg
      961 gcttgaccat gaacacctta tgtccgcccc aaagaacgct gtcggcgttc agcctccagc
     1021 cgctcaccca ctgctgcgcc tcttgattgg tcaacagctc catgaagttt ttcggcacgc
     1081 tcgtgaagta gttgttggta ttggtaaaat tgctggcgct gtggatgccc acgatgaagc
     1141 cgtccctagt gctcaccaat gggctgccgc attggccatc cttcgtctga atccagtgct
     1201 tccagaagat gccgtcgctg ctggggaagg tgcagctggt atcagagacc atgctgctca
     1261 tggacttggt ctggaagttg gtggtcacca ggcagattcg ctcctccctt tgtggctccc
     1321 tgaacttcag tttctgggga aaggggggga agtccttggg catcctgatg ataatcatgt
     1381 ccctgccgtc gatcaggtgc tgttgcaggg tggttgtgtt cttcactttg aacacgccgt
     1441 gcaggctttg caccagcagt gtgccgttat tcctcctgaa cagatgcttg ttggtgatga
     1501 tgaaggggcc gaagccgatg ccgtacagac tagtggtgtg accgtcgctc tcgttggtca
     1561 ggtgacagat ggtgctggag atcgggttgt agtccctcgg gcccttgaaa aggctctcgc
     1621 cagggccggg attctcctcc acgtcaccgc atgttagaag acttcctctg ccctctccgc
     1681 ttccggagtg ataaaaatag agttcttttg tgagcatgga aacaatacat ggaatctgtt
     1741 tctttgcatg aaaagtagcg tcaaaggaaa aggactcaaa ttctgttgcc acctttcggt
     1801 taacccgggt aagaatgtgc ataaattcaa gcttgtcggc atactgtttc agcatggcac
     1861 aaagcgactg gatgaaccag gagccatcct ttgaatttcg ccaagaataa taaccaggtg
     1921 ctgtggagta tgcatacaag aagtcggcct ccactggtat tttatgacac gccatgtcat
     1981 catcaacacc actctggaag tagagatttt ctgtctcaat gccacagtcc agttctgtac
     2041 cacggcaggc ctgaataatg aaaagtttgg gttttccagt tagacttcta caacgatccc
     2101 ctctgaaaaa gtttgttatt tttttcaggt caacaggtcc atttgttcca aaaattattc
     2161 cttcttcacc atggctcaga agcacacaaa caaaactgct ccttttgctg tgatcttctt
     2221 tagaaacatc acgcatcaat tccacaattt cttcacgtgt aagatcattt ttattcctga
     2281 cttcatattt caagtttctg aatgtttccc tgaggtttgc tgcatcgaca tctgtaccag
     2341 accgagatgt cattccagtg cttttatgaa aattcttatt attaattatt atacataaac
     2401 ccatctcagg ataatccatt ttataactgt tgtccaggga tattccagag tccattgatt
     2461 cgcttccatg tatgatcttt ggttccaaat ttttaatgga ttttgaatcc actgagtttt
     2521 cagtgttctc catgctagct agttggcgcg ccaacctggc cggccttgat aacttcgtat
     2581 aatgtatgct atacgaagtt atccgaatcg caataacttc gtataaagta tcctatacga
     2641 agttatcgaa gatcatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa
     2701 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     2761 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     2821 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     2881 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     2941 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     3001 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     3061 ggaaatcatc gtcctttcct tggctgctcg cctatgttgc cacctggatt ctgcgcggga
     3121 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     3181 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     3241 tttgggccgc ctccccgcat cgataccgag cgctgctcga gagatcgatc tgcctcgact
     3301 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     3361 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     3421 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     3481 gaagacaata gcaggcatgc tggggacacg tgcggaccga gcggccgcag gaacccctag
     3541 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     3601 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct
     3661 gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     3721 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3781 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3841 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3901 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     3961 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     4021 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     4081 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     4141 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt
     4201 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     4261 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     4321 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     4381 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     4441 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     4501 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     4561 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     4621 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     4681 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     4741 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     4801 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     4861 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     4921 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     4981 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     5041 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     5101 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     5161 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     5221 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     5281 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     5341 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     5401 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     5461 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     5521 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     5581 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     5641 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     5701 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     5761 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     5821 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     5881 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     5941 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6001 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     6061 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     6121 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     6181 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     6241 cttttgctgg ccttttgctc acatgt
//
