LOCUS       Exported                7212 bp ds-DNA     circular SYN 08-FEB-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7212)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Feb 8, 2022 from SnapGene 4.3.11
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7212
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     promoter        242..1419
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          473..1410
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1401..1424
                     /label=Seq-For
     protein_bind    1450..1483
                     /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."
     protein_bind    1534..1567
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             complement(1585..2301)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1627..1648)
                     /label=EGFP-C
     CDS             complement(2302..2358)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     misc_feature    complement(2359..2367)
                     /label=GSG
                     /note="Increase P2A activity?"
     misc_feature    complement(2368..2403)
                     /label=NES
                     /note="Nuclear Exporting Sequence - cytosolic targeting"
     CDS             complement(2404..3150)
                     /codon_start=1
                     /product="soybean ascorbate peroxidase, engineered to serve
                     as a monomeric tag for electron microscopy and live-cell 
                     proteomics (Lam et al., 2015)"
                     /label=APEX2
                     /note="more active than the original APEX due to the A134P 
                     mutation"
                     /translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
                     FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
                     EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
                     IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
                     YAADEDAFFADYAEAHQKLSELGFADA"
     primer_bind     2441..2461
                     /label=sq1808_APEX-C1
     primer_bind     2921..2946
                     /label=sq1811_APEX-N4
     primer_bind     complement(3112..3133)
                     /label=APEX-N
     CDS             complement(3151..3174)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    complement(3197..3230)
                     /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."
     protein_bind    complement(3281..3314)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    3339..3927
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3392..3412)
                     /label=sq1801_WPRE-R
     CDS             complement(3810..3821)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3959..4435
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     repeat_region   4475..4615
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4690..5145
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        5427..5531
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5532..6392
                     /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"
     rep_origin      6563..7151
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt
      181 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat
      241 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
      301 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
      361 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
      421 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
      481 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
      541 tacttccact ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      601 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      661 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      721 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      781 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      841 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      901 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      961 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     1021 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
     1081 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     1141 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     1201 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1261 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1321 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1381 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc
     1441 gactccggaa taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga
     1501 ttgtagctgc tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg
     1561 aagttatggc gcgccttaac cggtcttgta cagctcgtcc atgccgagag tgatcccggc
     1621 ggcggtcacg aactccagca ggaccatgtg atcgcgcttc tcgttggggt ctttgctcag
     1681 ggcggactgg gtgctcaggt agtggttgtc gggcagcagc acggggccgt cgccgatggg
     1741 ggtgttctgc tggtagtggt cggcgagctg cacgctgccg tcctcgatgt tgtggcggat
     1801 cttgaagttc accttgatgc cgttcttctg cttgtcggcc atgatataga cgttgtggct
     1861 gttgtagttg tactccagct tgtgccccag gatgttgccg tcctccttga agtcgatgcc
     1921 cttcagctcg atgcggttca ccagggtgtc gccctcgaac ttcacctcgg cgcgggtctt
     1981 gtagttgccg tcgtccttga agaagatggt gcgctcctgg acgtagcctt cgggcatggc
     2041 ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag cggctgaagc actgcacgcc
     2101 gtaggtcagg gtggtcacga gggtgggcca gggcacgggc agcttgccgg tggtgcagat
     2161 gaacttcagg gtcagcttgc cgtaggtggc atcgccctcg ccctcgccgg acacgctgaa
     2221 cttgtggccg tttacgtcgc cgtccagctc gaccaggatg ggcaccaccc cggtgaacag
     2281 ctcctcgccc ttgctcacca taggaccggg gttttcttcc acgtctcctg cttgctttaa
     2341 cagagagaag ttcgtggctc cggatccgtc cagggtcagg cgctccaggg gaggcagctg
     2401 cagggcatca gcaaacccaa gctcggaaag cttttggtga gcctcagcgt aatcagcaaa
     2461 gaaggcatct tcgtccgctg catatttgtc aacgagaggg cggaatacag ggtcagacaa
     2521 aagagccttg tcagaaggta gctgaaggag accttccttc tcaccactca acaactccgt
     2581 gaagtatgag ttgtcgaaaa taagaggatt agaggtccag ggaccctcaa atccagaacg
     2641 ctccttgtgt gcagctccaa tagtgtgacc cccagataga gcaacgatat cttggtcagt
     2701 aagccccata gctttgccaa acacatctct caaatggtca gaacccttag tgggatcggg
     2761 caagcgaccc tctggtggtg gctcaggctt gtcctctctt ccagggtgga atggaacctt
     2821 aggtccaccc gtgacctcaa cggcaacaac gccagccaac tggtagaaat cggcgtagct
     2881 caaaataggg aactccgcct tgagtggctc caaaagccta acagcgatgt caagaccgtt
     2941 gttagcgctg tgagccagtt cggcagggtg cttgatggtt ccgaagggtc caccggtctt
     3001 cgtgcccttg tcaaaggttc cagcagagtg gaatgccaaa cggagcatta gaggagcgca
     3061 tctcttctca gcgatgaagc ctctgagctt cttcttcgcc ttctcaacgg cgtcctggta
     3121 atcagcactc acagttgggt aagactttcc cttatcgtcg tcatccttgt agtccatcgg
     3181 cggcgcggtg gctagcataa cttcgtataa agtatcctat acgaagttat ttgccttaac
     3241 ccagaaatta tcactgttat tctttagaat ggtgcaaaga ataacttcgt ataatgtatg
     3301 ctatacgaag ttatgaattc gatatcaagc ttatcgataa tcaacctctg gattacaaaa
     3361 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     3421 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     3481 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     3541 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     3601 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     3661 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     3721 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctatgttgcc acctggattc
     3781 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     3841 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     3901 ggatctccct ttgggccgcc tccccgcatc gataccgagc gctgctcgag agatctacgg
     3961 gtggcatccc tgtgacccct ccccagtgcc tctcctggcc ctggaagttg ccactccagt
     4021 gcccaccagc cttgtcctaa taaaattaag ttgcatcatt ttgtctgact aggtgtcctt
     4081 ctataatatt atggggtgga ggggggtggt atggagcaag gggcaagttg ggaagacaac
     4141 ctgtagggcc tgcggggtct attgggaacc aagctggagt gcagtggcac aatcttggct
     4201 cactgcaatc tccgcctcct gggttcaagc gattctcctg cctcagcctc ccgagttgtt
     4261 gggattccag gcatgcatga ccaggctcag ctaatttttg tttttttggt agagacgggg
     4321 tttcaccata ttggccaggc tggtctccaa ctcctaatct caggtgatct acccaccttg
     4381 gcctcccaaa ttgctgggat tacaggcgtg aaccactgct cccttccctg tccttctgat
     4441 tttgtaggta accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc
     4501 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
     4561 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc
     4621 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc
     4681 aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     4741 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     4801 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     4861 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac
     4921 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     4981 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg ggctattctt
     5041 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     5101 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca
     5161 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     5221 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     5281 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     5341 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     5401 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     5461 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     5521 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     5581 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     5641 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     5701 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     5761 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     5821 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     5881 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     5941 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     6001 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     6061 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     6121 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     6181 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     6241 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     6301 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     6361 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     6421 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     6481 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6541 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     6601 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     6661 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     6721 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     6781 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     6841 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     6901 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     6961 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     7021 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     7081 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     7141 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     7201 ttgctcacat gt
//
