LOCUS       Exported                6833 bp ds-DNA     circular SYN 01-OCT-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 6833)
  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..6833
                     /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     912..936
                     /label=Primer 1
     regulatory      914..920
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             917..1633
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1613..1633)
                     /label=ps271
                     /note="WTHN hM3Dq F bsrg1"
     primer_bind     1613..1632
                     /label=PS310
                     /note="3p mCherry out"
     CDS             1655..1720
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus 
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     misc_feature    1706..3097
                     /label=Tet TOX (reverse)
                     /note="Tet TOX (reverse)"
     primer_bind     complement(3080..3107)
                     /label=Primer 2
     protein_bind    complement(3136..3169)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    complement(3180..3213)
                     /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    3237..3825
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3290..3310)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     misc_feature    complement(3294..3313)
                     /label=Feature 21
     primer_bind     complement(3294..3313)
                     /label=ps125
                     /note="Ai65 PCS seq R1"
     primer_bind     complement(3312..3331)
                     /label=ps145
                     /note="Rosa26TdT (007909) Mutant Reverse (WPRE)"
     primer_bind     complement(3330..3353)
                     /label=ps126
                     /note="Ai65 PCS seq R2"
     primer_bind     complement(3404..3423)
                     /label=ps178
                     /note="P18 R CRE-dsGFP deepseq"
     primer_bind     complement(3564..3579)
                     /label=ps124
                     /note="R Ai65 MluI test"
     CDS             complement(3708..3719)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3860..3877)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3866..4073
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3882..3901)
                     /label=ps176
                     /note="P1 R CRE-dsGFP deepseq"
     repeat_region   4096..4236
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4096..4225
                     /label=AAV2 ITR
     primer_bind     complement(4165..4175)
                     /label=ps211
                     /note="vwa5a 5pF1,use w/R2 (214) 493bp"
     rep_origin      4311..4766
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4398..4417)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4608..4629
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4783..4802)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4902..4924
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4962..4980)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5048..5152
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5153..6013
                     /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(5371..5390)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6184..6772
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6673..6692
                     /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 tgtacgatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
      961 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     1021 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     1081 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     1141 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     1201 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     1261 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     1321 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     1381 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     1441 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     1501 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     1561 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     1621 cgagctgtac aagggatcca aaattgtcgc tcctgtcaaa caaactctta actttgattt
     1681 actcaaactg gctggggatg tagaaagcaa tccaggtcca gaattcatgc cgatcaccat
     1741 caacaacttc cgttactccg acccggttaa caacgacacc atcatcatga tggaaccgcc
     1801 gtactgcaaa ggtctagaca tctactacaa agccttcaaa atcaccgacc gtatctggat
     1861 cgttccggaa cgttacgaat ttggtaccaa accggaagac ttcaacccgc cgtcctccct
     1921 gatcgaaggt gcttccgaat actacgaccc gaactacctg cgtaccgact ccgacaaaga
     1981 ccgtttcctg cagaccatgg ttaaactgtt caaccgtatc aaaaacaacg ttgctggtga
     2041 agctctgctg gacaaaatca tcaacgctat cccgtacctg ggtaactcct actccctgct
     2101 ggacaaattc gacaccaact ccaactccgt ttccttcaac ctgctggaac aggatccgtc
     2161 cggtgctacc accaaatccg ctatgctgac caacctgatc atcttcggtc cgggtccggt
     2221 tctgaacaaa aacgaagttc gtggtatcgt tctgcgtgtt gacaacaaaa actacttccc
     2281 gtgccgtgac ggtttcggtt ccatcatgca gatggctttc tgcccggaat acgtaccgac
     2341 cttcgacaac gttatcgaaa acatcacctc cctgaccatc ggtaaatcca aatacttcca
     2401 ggacccggct ctgctgctga tgcacgaact gatccacgtt ctgcacggtc tgtacggcat
     2461 gcaggtttcc tcccacgaaa tcatcccgtc caaacaggaa atctacatgc agcacaccta
     2521 cccgatctcc gctgaagagc tcttcacctt cggtggtcag gacgctaacc tgatctccat
     2581 cgatatcaaa aacgacctgt acgaaaaaac cctgaacgac tacaaagcta tcgctaacaa
     2641 actgtcccag gttacctcct gcaacgaccc gaacatcgac atcgactcct acaaacagat
     2701 ctatcagcag aagtatcagt ttgataagga ttccaacggc cagtatatag tgaacgagga
     2761 taagtttcag atactgtata actccataat gtatggcttt actgagatag agctgggcaa
     2821 gaagtttaac ataaagactc gcttaagcta tttttccatg aaccacgatc ctgtgaagat
     2881 acctaacctg ctggatgata ctatatataa cgatactgag ggctttaaca tagagtccaa
     2941 ggatctgaag tccgagtata agggccagaa catgagggtg aacactaacg cctttaggaa
     3001 cgtcgacggt tccggtctgg tttccaaact gatcggtctg tgcaaaaaaa tcatcccgcc
     3061 gaccaacatc cgtgaaaacc tgtacaaccg taccgcttaa gctagctagt tggcgcgcca
     3121 acctggccgg ccttgataac ttcgtataat gtatgctata cgaagttatc cgaatcgcaa
     3181 taacttcgta taaagtatcc tatacgaagt tatcgaagat catcaagctt atcgataatc
     3241 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     3301 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
     3361 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
     3421 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
     3481 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
     3541 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
     3601 gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct
     3661 atgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
     3721 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc
     3781 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgagcgc
     3841 tgctcgagag atcgatctgc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     3901 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     3961 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     4021 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggacacgtgc
     4081 ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     4141 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
     4201 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc
     4261 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg
     4321 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     4381 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     4441 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     4501 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
     4561 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     4621 ccaaactgga acaacactca accctatctc gggctattct tttgatttat aagggatttt
     4681 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     4741 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
     4801 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     4861 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     4921 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     4981 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     5041 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     5101 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     5161 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     5221 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     5281 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     5341 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     5401 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     5461 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     5521 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     5581 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     5641 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     5701 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     5761 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     5821 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     5881 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     5941 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     6001 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     6061 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     6121 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     6181 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     6241 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     6301 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     6361 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     6421 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     6481 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     6541 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     6601 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     6661 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     6721 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     6781 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
