LOCUS       Exported                6298 bp ds-DNA     circular SYN 16-APR-2020
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6298)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Apr 17, 2020 from SnapGene 5.0.7
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6298
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        228..607
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        608..811
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        856..874
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             900..977
                     /codon_start=1
                     /label=CD4 signal sequence
                     /translation="MCRAISLRRLLLLLLQLSQLLAVTQG"
     CDS             978..1043
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1048..1322
                     /codon_start=3
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTR"
     CDS             1323..1376
                     /codon_start=1
                     /label=Linker
                     /translation="GGSGGSGGSGGSGGSGGS"
     CDS             1383..1445
                     /codon_start=1
                     /label=CD28 TMD
                     /translation="LVVVAGVLFCYGLLVTVALCV"
     CDS             1446..1463
                     /codon_start=1
                     /label=Linker
                     /translation="GGGSGG"
     CDS             1470..1823
                     /codon_start=1
                     /label=CTEVp-119
                     /translation="KSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFI
                     VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPE
                     EPFQPVKEATQLMN"
     CDS             1824..1835
                     /codon_start=1
                     /label=Linker
                     /translation="LKGS"
     CDS             1836..1856
                     /codon_start=1
                     /label=CS(M)
                     /translation="ENLYFQM"
     CDS             1857..1874
                     /codon_start=1
                     /label=Linker
                     /translation="GGGSGG"
     CDS             1875..2876
                     /codon_start=1
                     /product="tetracycline-controlled transactivator, 
                     comprising a fusion of the tetracycline repressor TetR with
                     the C-terminal activation domain of herpes simplex virus 
                     VP16"
                     /label=tTA
                     /note="In the Tet-Off(R) system, tTA binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTI
                     EGLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHA
                     DALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYG"
     polyA_signal    2930..3154
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3200..3628
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     rep_origin      3698..3833
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3967..4088
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4137..4153)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4161..4177
                     /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)."
     promoter        complement(4185..4215)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4230..4251
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4539..5124)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5295..6155)
                     /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"
     promoter        complement(6156..6260)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata
       61 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
      121 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt
      181 taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt
      241 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
      301 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
      361 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
      421 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      481 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
      541 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
      601 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
      661 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
      721 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
      781 tgtacggtgg gaggtctata taagcagagc tctctggcta actagagaac ccactgctta
      841 ctggcttatc gaaattaata cgactcacta tagggagtcc caagctggct agcgccatca
      901 tgtgccgagc catctctctt aggcgcttgc tgctgctgct gctgcagctg tcacaactcc
      961 tagctgtcac tcaaggggat tacaaggatc acgatgggga ttacaaagat catgacattg
     1021 actacaagga tgacgatgat aagatgctcg agatgtggca tgaaggcctg gaagaggcat
     1081 ctcgtttgta ctttggggaa aggaacgtga aaggcatgtt tgaggtgctg gagcccttgc
     1141 atgctatgat ggaacggggc ccccagactc tgaaggaaac atcctttaat caggcctatg
     1201 gtcgagattt aatggaggcc caagagtggt gcaggaagta catgaaatca gggaatgtca
     1261 aggacctcct ccaagcctgg gacctctatt atcatgtgtt ccgacgaatc tcaaagacta
     1321 gaggaggatc tggaggctct gggggttcag gcggatcagg aggcagtggc gggtcaaccg
     1381 gtcttgtcgt cgttgcaggg gtgctcttct gttatggtct gttggtgaca gtcgcactgt
     1441 gtgtaggagg cggtagcgga ggcggcgcca agagcatgtc tagcatggtg tcagacacta
     1501 gttgcacatt cccttcatct gatggcatat tctggaagca ttggattcaa accaaggatg
     1561 ggcagtgtgg cagtccatta gtatcaacta gagatgggtt cattgttggt atacactcag
     1621 catcgaattt caccaacaca aacaattatt tcacaagcgt gccgaaaaac ttcatggaat
     1681 tgttgacaaa tcaggaggcg cagcagtggg ttagtggttg gcgattaaat gctgactcag
     1741 tattgtgggg gggccataaa gttttcatgg tgaaacctga agagcctttt cagccagtta
     1801 aggaagcgac tcaactcatg aatcttaagg gctcggagaa cctgtatttt cagatggggg
     1861 gaggcagcgg gggcatgtct agattagata aaagtaaagt gattaacagc gcattagagc
     1921 tgcttaatga ggtcggaatc gaaggtttaa caacccgtaa actcgcccag aagctaggtg
     1981 tagagcagcc tacattgtat tggcatgtaa aaaataagcg ggctttgctc gacgccttag
     2041 ccattgagat gttagatagg caccatactc acttttgccc tttagaaggg gaaagctggc
     2101 aagatttttt acgtaataac gctaaaagtt ttagatgtgc tttactaagt catcgcgatg
     2161 gagcaaaagt acatttaggt acacggccta cagaaaaaca gtatgaaact ctcgaaaatc
     2221 aattagcctt tttatgccaa caaggttttt cactagagaa tgcattatat gcactcagcg
     2281 ctgtggggca ttttacttta ggttgcgtat tggaagatca agagcatcaa gtcgctaaag
     2341 aagaaaggga aacacctact actgatagta tgccgccatt attacgacaa gctatcgaat
     2401 tatttgatca ccaaggtgca gagccagcct tcttattcgg ccttgaattg atcatatgcg
     2461 gattagaaaa acaacttaaa tgtgaaagtg ggtccgcgta cagccgcgcg cgtacgaaaa
     2521 acaattacgg gtctaccatc gagggcctgc tcgatctccc ggacgacgac gcccccgaag
     2581 aggcggggct ggcggctccg cgcctgtcct ttctccccgc gggacacacg cgcagactgt
     2641 cgacggcccc cccgaccgat gtcagcctgg gggacgagct ccacttagac ggcgaggacg
     2701 tggcgatggc gcatgccgac gcgctagacg atttcgatct ggacatgttg ggggacgggg
     2761 attccccggg tccgggattt accccccacg actccgcccc ctacggcgct ctggatatgg
     2821 ccgacttcga gtttgagcag atgtttaccg atgcccttgg aattgacgag tacggttagg
     2881 cggccgctcg agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct
     2941 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     3001 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     3061 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagtcaat
     3121 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     3181 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     3241 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     3301 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     3361 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     3421 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     3481 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     3541 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     3601 ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttaggctcaa
     3661 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     3721 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     3781 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     3841 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga ggttgggctt cggaatcgtt
     3901 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     3961 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     4021 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     4081 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     4141 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4201 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4261 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4321 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4381 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4441 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4501 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4561 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4621 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4681 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4741 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4801 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4861 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4921 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4981 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5041 tcttgatccg gcaaacaaac caccgctggt agcggttttt ttgtttgcaa gcagcagatt
     5101 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5161 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5221 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5281 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5341 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5401 ttaccatctg gccccagtgc tgcaatgata ccgcgagatc cacgctcacc ggctccagat
     5461 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     5521 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     5581 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     5641 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     5701 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     5761 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     5821 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     5881 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     5941 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6001 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6061 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6121 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6181 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6241 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggat
//
