LOCUS       62056_21425        7306 bp DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7306)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7306
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    208..241
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        251..629
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     CDS             775..1491
                     /codon_start=1
                     /label=EGFP
                     /note="enhanced GFP"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             1540..1563
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     polyA_signal    1581..1805
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1901..2112
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2125..2393
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human
                     T-cell leukemia virus (HTLV) type 1"
     CDS             2427..3170
                     /codon_start=1
                     /label=Tet-On(R) 3G
                     /note="modified rtTA protein that binds tightly to
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLSADALDDFDLDMLPG"
     CDS             3171..3224
                     /codon_start=1
                     /label=T2A
                     /note="2A peptide from Thosea asigna virus capsid protein"
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3231..3827
                     /codon_start=1
                     /label=PuroR
                     /note="puromycin N-acetyltransferase"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     polyA_signal    3950..4071
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(4350..4368)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4378..4394)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      4536..4991
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        5017..5121
                     /label=AmpR promoter
     CDS             5122..5979
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      6153..6741
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    7029..7050
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        7065..7095
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7103..7119
                     /label=lac operator
                     /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     7127..7143
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        7164..7182
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 agtcgccgct tggagctccc gtgaggcgtg cttgtcaatg cggtaagtgt cactgatttt
       61 gaactataac gaccgcgtga gtcaaaatga cgcatgatta tcttttacgt gacttttaag
      121 atttaactca tacgataatt atattgttat ttcatgttct acttacgtga taacttatta
      181 tatatatatt ttcttgttat agatatcata acttcgtata atgtatgcta tacgaagtta
      241 tgcggccctc gagtttactc cctatcagtg atagagaacg tatgaagagt ttactcccta
      301 tcagtgatag agaacgtatg cagactttac tccctatcag tgatagagaa cgtataagga
      361 gtttactccc tatcagtgat agagaacgta tgaccagttt actccctatc agtgatagag
      421 aacgtatcta cagtttactc cctatcagtg atagagaacg tatatccagt ttactcccta
      481 tcagtgatag agaacgtata agctttaggc gtgtacggtg ggcgcctata aaagcagagc
      541 tcgtttagtg aaccgtcaga tcgcctggag caattccaca acacttttgt cttataccaa
      601 ctttccgtac cacttcctac cctcgtaaag tcgacaccgg ggcccagatc tatcgatcgg
      661 ccggatatca cgcgtcatat ggctagcctg cagggatccg gtagatcggg tgaattcgcc
      721 accaacttca gcctgctgaa gcaggccggc gacgtggagg agaaccccgg ccccatggtg
      781 tctaagggcg aagagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
      841 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
      901 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
      961 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     1021 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     1081 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     1141 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     1201 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     1261 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     1321 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1381 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1441 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa ggtcgacgac
     1501 tacaaagacc atgacggtga ttataaagat catgacatcg actacaagga tgacgatgac
     1561 aagtagtcta gcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     1621 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     1681 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     1741 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     1801 tatggcttct gaggcggact agagcgatcc ctaggctagg cttttgcaaa gctagcagct
     1861 gaatctaagt ggatctgcga tcgctccggt gcccgtcagt gggcagagcg cacatcgccc
     1921 acagtccccg agaagttggg gggaggggtc ggcaattgaa cgggtgccta gagaaggtgg
     1981 cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg
     2041 ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc
     2101 gccagaacac agctgaagca tcgaggggct cgcatctctc cttcacgcgc ccgccgccct
     2161 acctgaggcc gccatccacg ccggttgagt cgcgttctgc cgcctcccgc ctgtggtgcc
     2221 tcctgaactg cgtccgccgt ctaggtaagt ttaaagctca ggtcgagacc gggcctttgt
     2281 ccggcgctcc cttggagcct acctagactc agccggctct ccacgctttg cctgaccctg
     2341 cttgctcaac tctacgtctt tgtttcgttt tctgttctgc gccgttacag atccaagctg
     2401 tgaccggcgc ctactctaga gccgccatgt ctagactgga caagagcaaa gtcataaact
     2461 ctgctctgga attactcaat ggagtcggta tcgaaggcct gacgacaagg aaactcgctc
     2521 aaaagctggg agttgagcag cctaccctgt actggcacgt gaagaacaag cgggccctgc
     2581 tcgatgccct gccaatcgag atgctggaca ggcatcatac ccactcctgc cccctggaag
     2641 gcgagtcatg gcaagacttt ctgcggaaca acgccaagtc ataccgctgt gctctcctct
     2701 cacatcgcga cggggctaaa gtgcatctcg gcacccgccc aacagagaaa cagtacgaaa
     2761 ccctggaaaa tcagctcgcg ttcctgtgtc agcaaggctt ctccctggag aacgcactgt
     2821 acgctctgtc cgccgtgggc cactttacac tgggctgcgt attggaggaa caggagcatc
     2881 aagtagcaaa agaggaaaga gagacaccta ccaccgattc tatgccccca cttctgaaac
     2941 aagcaattga gctgttcgac cggcagggag ccgaacctgc cttccttttc ggcctggaac
     3001 taatcatatg tggcctggag aaacagctaa agtgcgaaag cggcgggccg accgacgccc
     3061 ttgacgattt tgacttagac atgctcccag ccgatgccct tgacgacttt gaccttgata
     3121 tgctgtctgc tgacgctctt gacgattttg accttgacat gctccccggg gagggcagag
     3181 gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccctcttaag atgaccgagt
     3241 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg
     3301 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg
     3361 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     3421 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag
     3481 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     3541 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     3601 tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     3661 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     3721 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     3781 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga gcggccgcga
     3841 ctctagtcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc
     3901 cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgta acttgtttat
     3961 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4021 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt aggtacaagt
     4081 aggcgcgtcg agataacttc gtataatgta tgctatacga agttatgttt tgttacttta
     4141 tagaagaaat tttgagtttt tgtttttttt taataaataa ataaacataa ataaattgtt
     4201 tgttgaattt attattagta tgtaagtgta aatataataa aacttaatat ctattcaaat
     4261 taataaataa acgtcaattt tacgcatgat tatctttaac gtacgtcaca atatgattat
     4321 ctttctaggg ttaaagagct ccaattcgcc ctatagtgag tcgtattacg cgcgctcact
     4381 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     4441 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     4501 ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag
     4561 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     4621 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     4681 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     4741 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     4801 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     4861 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     4921 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     4981 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     5041 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     5101 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     5161 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     5221 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     5281 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     5341 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     5401 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     5461 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     5521 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     5581 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     5641 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     5701 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     5761 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     5821 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     5881 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     5941 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     6001 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     6061 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     6121 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     6181 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     6241 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     6301 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     6361 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     6421 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     6481 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     6541 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     6601 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     6661 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     6721 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     6781 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     6841 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     6901 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
     6961 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     7021 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     7081 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     7141 gaccatgatt acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctgggtacc
     7201 ttaaccctag aaagatagtc tgcgtaaaat tgacgcatgc attcttgaaa tattgctctc
     7261 tctttctaaa tagcgcgaat ccgtcgctgt gcatttagga catctc
//
