LOCUS       Exported                6819 bp ds-DNA     circular SYN 06-FEB-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 6819)
  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..6819
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     repeat_region   12..141
                     /label=AAV2 ITR
     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"
     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."
     misc_feature    900..2291
                     /label=Tet TOX (reverse)
                     /note="Tet TOX (reverse)"
     CDS             complement(2277..2342)
                     /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"
     CDS             complement(2364..3080)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     regulatory      3077..3086
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(3122..3155)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    complement(3166..3199)
                     /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    3223..3811
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3276..3296)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3694..3705)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3846..3863)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3852..4059
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4082..4211
                     /label=AAV2 ITR
     rep_origin      4297..4752
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4384..4403)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4594..4615
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4769..4788)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4888..4910
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4948..4966)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5034..5138
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5139..5999
                     /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(5357..5376)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6170..6758
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6659..6678
                     /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 gggcccttaa
      901 gcggtacggt tgtacaggtt ttcacggatg ttggtcggcg ggatgatttt tttgcacaga
      961 ccgatcagtt tggaaaccag accggaaccg tcgacgttcc taaaggcgtt agtgttcacc
     1021 ctcatgttct ggcccttata ctcggacttc agatccttgg actctatgtt aaagccctca
     1081 gtatcgttat atatagtatc atccagcagg ttaggtatct tcacaggatc gtggttcatg
     1141 gaaaaatagc ttaagcgagt ctttatgtta aacttcttgc ccagctctat ctcagtaaag
     1201 ccatacatta tggagttata cagtatctga aacttatcct cgttcactat atactggccg
     1261 ttggaatcct tatcaaactg atacttctgc tgatagatct gtttgtagga gtcgatgtcg
     1321 atgttcgggt cgttgcagga ggtaacctgg gacagtttgt tagcgatagc tttgtagtcg
     1381 ttcagggttt tttcgtacag gtcgtttttg atatcgatgg agatcaggtt agcgtcctga
     1441 ccaccgaagg tgaagagctc ttcagcggag atcgggtagg tgtgctgcat gtagatttcc
     1501 tgtttggacg ggatgatttc gtgggaggaa acctgcatgc cgtacagacc gtgcagaacg
     1561 tggatcagtt cgtgcatcag cagcagagcc gggtcctgga agtatttgga tttaccgatg
     1621 gtcagggagg tgatgttttc gataacgttg tcgaaggtcg gtacgtattc cgggcagaaa
     1681 gccatctgca tgatggaacc gaaaccgtca cggcacggga agtagttttt gttgtcaaca
     1741 cgcagaacga taccacgaac ttcgtttttg ttcagaaccg gacccggacc gaagatgatc
     1801 aggttggtca gcatagcgga tttggtggta gcaccggacg gatcctgttc cagcaggttg
     1861 aaggaaacgg agttggagtt ggtgtcgaat ttgtccagca gggagtagga gttacccagg
     1921 tacgggatag cgttgatgat tttgtccagc agagcttcac cagcaacgtt gtttttgata
     1981 cggttgaaca gtttaaccat ggtctgcagg aaacggtctt tgtcggagtc ggtacgcagg
     2041 tagttcgggt cgtagtattc ggaagcacct tcgatcaggg aggacggcgg gttgaagtct
     2101 tccggtttgg taccaaattc gtaacgttcc ggaacgatcc agatacggtc ggtgattttg
     2161 aaggctttgt agtagatgtc tagacctttg cagtacggcg gttccatcat gatgatggtg
     2221 tcgttgttaa ccgggtcgga gtaacggaag ttgttgatgg tgatcggcat gaattctgga
     2281 cctggattgc tttctacatc cccagccagt ttgagtaaat caaagttaag agtttgtttg
     2341 acaggagcga caattttgga tcccttgtac agctcgtcca tgccgagagt gatcccggcg
     2401 gcggtcacga actccagcag gaccatgtga tcgcgcttct cgttggggtc tttgctcagg
     2461 gcggactggg tgctcaggta gtggttgtcg ggcagcagca cggggccgtc gccgatgggg
     2521 gtgttctgct ggtagtggtc ggcgagctgc acgctgccgt cctcgatgtt gtggcggatc
     2581 ttgaagttca ccttgatgcc gttcttctgc ttgtcggcca tgatatagac gttgtggctg
     2641 ttgtagttgt actccagctt gtgccccagg atgttgccgt cctccttgaa gtcgatgccc
     2701 ttcagctcga tgcggttcac cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg
     2761 tagttgccgt cgtccttgaa gaagatggtg cgctcctgga cgtagccttc gggcatggcg
     2821 gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca ctgcacgccg
     2881 taggtcaggg tggtcacgag ggtgggccag ggcacgggca gcttgccggt ggtgcagatg
     2941 aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga cacgctgaac
     3001 ttgtggccgt ttacgtcgcc gtccagctcg accaggatgg gcaccacccc ggtgaacagc
     3061 tcctcgccct tgctcaccat ggtggcgcta gctagttggc gcgccaacct ggccggcctt
     3121 gataacttcg tataatgtat gctatacgaa gttatccgaa tcgcaataac ttcgtataaa
     3181 gtatcctata cgaagttatc gaagatcatc aagcttatcg ataatcaacc tctggattac
     3241 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     3301 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     3361 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     3421 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     3481 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     3541 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     3601 gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctatgt tgccacctgg
     3661 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     3721 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     3781 agtcggatct ccctttgggc cgcctccccg catcgatacc gagcgctgct cgagagatcg
     3841 atctgcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     3901 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     3961 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     4021 gggggaggat tgggaagaca atagcaggca tgctggggac acgtgcggac cgagcggccg
     4081 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     4141 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     4201 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     4261 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     4321 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     4381 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     4441 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     4501 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     4561 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     4621 cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct
     4681 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     4741 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     4801 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     4861 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     4921 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     4981 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     5041 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     5101 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     5161 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     5221 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     5281 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     5341 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     5401 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     5461 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     5521 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     5581 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     5641 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     5701 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     5761 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     5821 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     5881 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     5941 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     6001 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     6061 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     6121 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     6181 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     6241 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     6301 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     6361 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     6421 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     6481 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     6541 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     6601 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     6661 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     6721 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     6781 tacggttcct ggccttttgc tggccttttg ctcacatgt
//
