LOCUS       Exported                5498 bp DNA     circular SYN 20-JUN-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 5498)
  AUTHORS   Vahid Asimi
  TITLE     Direct Submission
  JOURNAL   Exported Jun 21, 2022 from SnapGene 6.0.5
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5498
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     protein_bind    106..127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        142..172
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    180..196
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             216..270
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDP"
     primer_bind     231..297
                     /label=Fragment 1.FOR
     misc_feature    233..270
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     misc_feature    271..1070
                     /label=5' Homology Region TRIM28
     primer_bind     complement(838..847)
                     /label=Fragment 3.REV
     primer_bind     1053..1091
                     /label=Fragment 2.FOR
     primer_bind     complement(1057..1092)
                     /label=Fragment 1.REV
     misc_feature    1057..1072
                     /label=sgRNA1
     regulatory      1071..1076
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    1071..1072
                     /label=to correct frame
     CDS             1073..1783
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     primer_bind     1080..1103
                     /label=VA_T28_repair_seq_f1
     primer_bind     complement(1124..1145)
                     /label=VA_T28_repair_seq_r1
     primer_bind     complement(1634..1653)
                     /label=VA_T28_repair_seq_r2
     primer_bind     1667..1686
                     /label=VA_T28_repair_seq_f2
     primer_bind     complement(1763..1803)
                     /label=Fragment 2.REV
     primer_bind     1764..1806
                     /label=Fragment 3.FOR
     misc_feature    1766..1785
                     /label=same in GS and P2A
     CDS             1793..1849
                     /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"
     CDS             1850..1876
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1880..1906
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(1914..1935)
                     /label=FKBP-V_R
     CDS             1916..2236
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=FKBP-V
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     misc_feature    2237..2281
                     /label=(GGGGS)x3
     primer_bind     2262..2295
                     /label=Fragment 4.FOR
     primer_bind     complement(2267..2301)
                     /label=Fragment 3.REV
     misc_feature    2282..2288
                     /label=sgRNA1
     misc_feature    2282
                     /label=to correct frame
     misc_feature    2283..3082
                     /label=3' Homology Region TRIM28
     primer_bind     2516..2535
                     /label=VA_T28_repair_seq_f3
     primer_bind     complement(2524..2543)
                     /label=VA_T28_repair_seq_r3
     primer_bind     complement(3066..3122)
                     /label=Fragment 4.REV
     CDS             3083..3351
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="VPSSNSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTD
                     RPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICSDAA"
     misc_feature    3083..3101
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(3102..3118)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3592..3696
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3697..4557
                     /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      4728..5316
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
       61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      181 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc
      241 atgcctgcag gtcgactcta gaggatcccc ccgagtttaa caataagaaa gtttgggtta
      301 agaccgcctc cacgttcggc gggaacttcc tggcaaagac cccctcaccc cctccacctg
      361 ggctccggcg ctttgctctg ccgtggcccc gcccaccctg tattgtcccg cccctcttcc
      421 ccgactatag gccaggctgc cgctgcagac gtcgccacgc ctgctgagga ggcggggcct
      481 aaggttcggc tctgcaggac ctgcgcgctc aagcacgcgg gttgccagcc tgggtgcacc
      541 gtgcggtggg catgggagac gtcaagcacg cactctgcgg gtgaattcct caccagcagc
      601 attgctactc tggatctaga ggggcggcag cgagtggaag ggagggccct ggcgcgtgcg
      661 taatctgcgc cccgccccca ggctgcggtg cccaacgcgc gtgcgcggcg tcgtcggcgc
      721 cagatatttc tgtcccgcct ccaggccgcg gctctttctg cgagcggcgc gcggtcaggc
      781 ggttgttcgc aagtcttgcg gcgcgagtgt ctcagaagcg agggaggagg cggcgctggt
      841 ggcggcgccg tcgtctgtac agaggcccgc agccagcggc ttggccgcat ctcgacagcg
      901 cctggggcgc ggcgggcgtc ggcccaggag acgcgtggcg gcgctcggcc tcgcggcatc
      961 ggcggctgcc tggccgttgg cggcgagcgc acttgcgcct gcgcagcggg ctccgtgccc
     1021 ctcctcccct gggcggcccc cccacccccc cggcggcgtg tgaatggcgg ccatggtgtc
     1081 taagggcgaa gagctgatca aggaaaatat gcgtatgaag gtggtcatgg aaggttcggt
     1141 caacggccac caattcaaat gcacaggtga aggagaaggc aatccgtaca tgggaactca
     1201 aaccatgagg atcaaagtca tcgagggagg acccctgcca tttgcctttg acattcttgc
     1261 cacgtcgttc atgtatggca gccgtacttt tatcaagtac ccgaaaggca ttcctgattt
     1321 ctttaaacag tcctttcctg agggttttac ttgggaaaga gttacgagat acgaagatgg
     1381 tggagtcgtc accgtcatgc aggacaccag ccttgaggat ggctgtctcg tttaccacgt
     1441 ccaagtcaga ggggtaaact ttccctccaa tggtcccgtg atgcagaaga agaccaaggg
     1501 ttgggagcct aatacagaga tgatgtatcc agcagatggt ggtctgaggg gatacactca
     1561 tatggcactg aaagttgatg gtggtggcca tctgtcttgc tctttcgtaa caacttacag
     1621 gtcaaaaaag accgtcggga acatcaagat gcccggtatc catgccgttg atcaccgcct
     1681 ggaaaggtta gaggaaagtg acaatgaaat gttcgtagta caacgcgaac acgcagttgc
     1741 caagttcgcc gggcttggtg gtgggatgga cgagctgtac aagggaagcg gagctactaa
     1801 cttcagcctg ctgaagcagg ctggagacgt ggaggagaac cctggacctt acccctacga
     1861 cgtgcccgac tacgccggct atccgtatga tgtcccggac tatgcaggaa gcggaggagt
     1921 gcaggtggaa accatctccc caggagacgg gcgcaccttc cccaagcgcg gccagacctg
     1981 cgtggtgcac tacaccggga tgcttgaaga tggaaagaaa gttgattcct cccgggacag
     2041 aaacaagccc tttaagttta tgctaggcaa gcaggaggtg atccgaggct gggaagaagg
     2101 ggttgcccag atgagtgtgg gtcagagagc caaactgact atatctccag attatgccta
     2161 tggtgccact gggcacccag gcatcatccc accacatgcc actctcgtct tcgatgtgga
     2221 gcttctaaaa ctggaaggtg gcggtggctc gggcggtggt gggtcgggtg gcggcggatc
     2281 tgcctcggcg gcagcgactg cagcggcctc ggccgcgacg gccgcctcgg cggcctctgg
     2341 tagcccaggg tcgggcgagg gctcggcggg cggtgagaag cgtccggctg cttcctcagc
     2401 cgcggcggcc tctgcagccg cgtcgtcccc tgcggggggc ggtggcgagg cgcaggagct
     2461 tctggagcac tgcggcgtgt gtcgcgagcg cctgcggccc gagcgggatc ctcggctgct
     2521 gccctgtcta cattcggcct gcagtgcctg cctgggcccc gctacacccg ccgcagcgaa
     2581 taattcgggg gatggcggct cggcgggcga cggcgctagt gagtactggg tggccaggtg
     2641 cccctccccc tcctcgcagc ccgtgctcgg gactgcgcct gtgcgagagt atgggggccc
     2701 gggtagggtt aagtaggcct gctggtttaa gagggggcgg ggaacgggtc ctggcctctg
     2761 ccaatgcccg ttaccaggtc tggacaccga ggtgcagaat gtgatgggag atgtcaagaa
     2821 atcagagggt gtgcatgcat tcccattttt atcttggcag tggtggattg tccagtgtgc
     2881 aaacagcagt gctactccaa agacatcgtg gagaattatt ttatgcgtga tagtggcagt
     2941 aaggcctctt ctgattccca ggatgctaac caggtatgtc ctgtgggaaa tcttggggaa
     3001 gagggaagcg tgaccttttc cagagcctct caccatcttc actagtttgg attgtatcct
     3061 tatgcggtct cagtgtctgg gagggtaccg agctcgaatt cactggccgt cgttttacaa
     3121 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     3181 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     3241 agcctgaatg gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     3301 tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     3361 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     3421 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     3481 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     3541 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     3601 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3661 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     3721 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     3781 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     3841 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     3901 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     3961 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     4021 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     4081 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     4141 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     4201 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     4261 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     4321 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     4381 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     4441 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     4501 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     4561 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     4621 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     4681 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4741 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4801 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4861 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4921 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4981 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     5041 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     5101 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     5161 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     5221 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     5281 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5341 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     5401 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     5461 accgagcgca gcgagtcagt gagcgaggaa gcggaaga
//
