LOCUS       62056_3405        7497 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 7497)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7497
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /label=AmpR promoter
     rep_origin      complement(131..586)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     728..744
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1382..1760
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     polyA_signal    1896..1944
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        2175..2386
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2399..2667
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human
                     T-cell leukemia virus (HTLV) type 1"
     CDS             2701..3444
                     /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             3445..3498
                     /codon_start=1
                     /label=T2A
                     /note="2A peptide from Thosea asigna virus capsid protein"
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3505..4101
                     /codon_start=1
                     /label=PuroR
                     /note="puromycin N-acetyltransferase"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     polyA_signal    4157..4278
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5476..5492)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5500..5516)
                     /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)."
     promoter        complement(5524..5554)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5569..5590)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(5878..6466)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6640..7497)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
      181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
      241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
      301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
      361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
      421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
      481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
      541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
      601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
      661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
      721 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg
      781 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg
      841 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg
      901 tacgttaaag ataatcatgc gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
      961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
     1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaaa caaaaactca
     1081 aaatttcttc tataaagtaa caaaactttt atgagggaca gccccccccc aaagccccca
     1141 gggatgtaat tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct
     1201 ccggtccggc gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg
     1261 gggaaggtgg cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag
     1321 acacctgggg ggatacgggg aaaaggcctc caaggcctac tagtcccttt cgtcttcact
     1381 cgagtttact ccctatcagt gatagagaac gtatgaagag tttactccct atcagtgata
     1441 gagaacgtat gcagacttta ctccctatca gtgatagaga acgtataagg agtttactcc
     1501 ctatcagtga tagagaacgt atgaccagtt tactccctat cagtgataga gaacgtatct
     1561 acagtttact ccctatcagt gatagagaac gtatatccag tttactccct atcagtgata
     1621 gagaacgtat aagctatagg cgtgtacggt gggcgcctat aaaagcagag ctcgtttagt
     1681 gaaccgtcag atcgcctgga gcaattccac aacacttttg tcttatacca actttccgta
     1741 ccacttccta ccctcgtaaa ggaagctttc tagagctagc gtcgacacgc gtgcggccgc
     1801 tgtacaatgg gtaccaccgg ttagtaatga gtttaaacgg gggaggctaa ctgaaacacg
     1861 gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac
     1921 gcacgggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg
     1981 tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac
     2041 cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc
     2101 ctgccatagc agatctgcga aatcggatcc agctgaatct aagtggatct gcgatcgctc
     2161 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
     2221 ggtcggcaat tgaacgggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
     2281 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
     2341 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacagctga agcatcgagg
     2401 ggctcgcatc tctccttcac gcgcccgccg ccctacctga ggccgccatc cacgccggtt
     2461 gagtcgcgtt ctgccgcctc ccgcctgtgg tgcctcctga actgcgtccg ccgtctaggt
     2521 aagtttaaag ctcaggtcga gaccgggcct ttgtccggcg ctcccttgga gcctacctag
     2581 actcagccgg ctctccacgc tttgcctgac cctgcttgct caactctacg tctttgtttc
     2641 gttttctgtt ctgcgccgtt acagatccaa gctgtgaccg gcgcctactc tagagccgcc
     2701 atgtctagac tggacaagag caaagtcata aactctgctc tggaattact caatggagtc
     2761 ggtatcgaag gcctgacgac aaggaaactc gctcaaaagc tgggagttga gcagcctacc
     2821 ctgtactggc acgtgaagaa caagcgggcc ctgctcgatg ccctgccaat cgagatgctg
     2881 gacaggcatc atacccactc ctgccccctg gaaggcgagt catggcaaga ctttctgcgg
     2941 aacaacgcca agtcataccg ctgtgctctc ctctcacatc gcgacggggc taaagtgcat
     3001 ctcggcaccc gcccaacaga gaaacagtac gaaaccctgg aaaatcagct cgcgttcctg
     3061 tgtcagcaag gcttctccct ggagaacgca ctgtacgctc tgtccgccgt gggccacttt
     3121 acactgggct gcgtattgga ggaacaggag catcaagtag caaaagagga aagagagaca
     3181 cctaccaccg attctatgcc cccacttctg aaacaagcaa ttgagctgtt cgaccggcag
     3241 ggagccgaac ctgccttcct tttcggcctg gaactaatca tatgtggcct ggagaaacag
     3301 ctaaagtgcg aaagcggcgg gccgaccgac gcccttgacg attttgactt agacatgctc
     3361 ccagccgatg cccttgacga ctttgacctt gatatgctgt ctgctgacgc tcttgacgat
     3421 tttgaccttg acatgctccc cggggagggc agaggaagtc ttctaacatg cggtgacgtg
     3481 gaggagaatc ccggccctct taagatgacc gagtacaagc ccacggtgcg cctcgccacc
     3541 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     3601 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     3661 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     3721 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     3781 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     3841 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc
     3901 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     3961 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     4021 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     4081 atgacccgca agcccggtgc ctgagaattc tacctacgct atgtggatac gctgctttaa
     4141 tgcctttgta tcagttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4201 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4261 actcatcaat gtatcttaag ctcatctggt ctcccttccg ggggacaaga catccctgtt
     4321 taatatttaa acagcagtgt tcccaaactg ggttcttata tcccttgctc tggtcaacca
     4381 ggttgcaggg tttcctgtcc tcacaggaac gaagtcccta aagaaacagt ggcagccagg
     4441 tttagccccg gaattgactg gattcctttt ttagggccca ttggtatggc tttttccccg
     4501 tatcccccca ggtgtctgca ggctcaaaga gcagcgagaa gcgttcagag gaaagcgatc
     4561 ccgtgccacc ttccccgtgc ccgggctgtc cccgcacgct gccggctcgg ggatgcgggg
     4621 ggagcgccgg accggagcgg agccccgggc ggctcgctgc tgccccctag cgggggagga
     4681 tctataacaa gaaaatatat atataataag ttatcacgta agtagaacat gaaataacaa
     4741 tataattatc gtatgagtta aatcttaaaa gtcacgtaaa agataatcat gcgtcatttt
     4801 gactcacgcg gtcgttatag ttcaaaatca gtgacactta ccgcattgac aagcacgcct
     4861 cacgggagct ccaagcggcg actgagatgt cctaaatgca cagcgacgga ttcgcgctat
     4921 ttagaaagag agagcaatat ttcaagaatg catgcgtcaa ttttacgcag actatctttc
     4981 tagggttaat ctagctgcat caggatcata tcgtcgggtc ttttttccgg ctcagtcatc
     5041 gcccaagctg gcgctatctg ggcatcgggg aggaagaagc ccgtgccttt tcccgcgagg
     5101 ttgaagcggc atggaaagag tttgccgagg atgactgctg ctgcattgac gttgagcgaa
     5161 aacgcacgtt taccatgatg attcgggaag gtgtggccat gcacgccttt aacggtgaac
     5221 tgttcgttca ggccacctgg gataccagtt cgtcgcggct tttccggaca cagttccgga
     5281 tggtcagccc gaagcgcatc agcaacccga acaataccgg cgacagccgg aactgccgtg
     5341 ccggtgtgca gattaatgac agcggtgcgg cgctgggata ttacgtcagc gaggacgggt
     5401 atcctggctg gatgccgcag aaatggacat ggataccccg tgagttaccc ggcgggcgcg
     5461 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     5521 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     5581 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5641 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     5701 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     5761 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5821 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5881 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5941 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     6001 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     6061 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     6121 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     6181 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     6241 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     6301 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     6361 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     6421 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     6481 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     6541 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     6601 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     6661 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     6721 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     6781 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6841 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6901 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     6961 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     7021 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     7081 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     7141 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     7201 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     7261 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     7321 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     7381 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     7441 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcat
//
