LOCUS       V040033                 6736 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V040033
VERSION     V040033
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     enhancer        235..614
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
     promoter        615..818
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /label="CMV promoter"
     promoter        863..881
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /label="T7 promoter"
     CDS             928..2172
                     /label="CRHR1(NM_004382)"
                     /note="CRHR1(NM_004382)"
                     /gene="CRHR1"
     CDS             2179..2202
                     /codon_start=1
                     /product="FLAG® epitope tag, followed by an enterokinase
                     cleavage site"
                     /transl_table=1
                     /translation="DYKDDDDK"
                     /label="FLAG"
     CDS             2209..2298
                     /codon_start=1
                     /product="two Strep-Tag®II moieties connected by a linker
                     for enhanced binding to Strep-Tactin®,&nbsp;an engineered
                     form of streptavidin (Schmidt et al., 2013)"
                     /transl_table=1
                     /translation="SA,WSHPQFEK,GGGSGGGSGGSA,WSHPQFEK"
                     /label="Twin-Strep-tag"
     polyA_signal    2336..2560
                     /note="bovine growth hormone polyadenylation signal"
                     /label="bGH poly(A) signal"
     rep_origin      2606..3034
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        3048..3377
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             3444..4238
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /note="confers resistance to neomycin, kanamycin, and G418
                     (Geneticin®)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /transl_table=1
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
                     /label="NeoR/KanR"
     polyA_signal    4412..4533
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(4582..4598)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    4606..4622
                     /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-β-D-thiogalactopyranoside (IPTG)."
                     /label="lac operator"
     promoter        complement(4630..4660)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    4675..4696
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(4984..5569)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(5740..6600)
                     /codon_start=1
                     /gene="bla"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /product="β-lactamase"
                     /transl_table=1
                     /translation="MSIQHFRVALIPFFAAFCLPVFA,HPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEY
                     SPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDR
                     WEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRS
                     ALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGA
                     SLIKHW*"
                     /label="AmpR"
     promoter        complement(6601..6705)
                     /gene="bla"
                     /label="AmpR promoter"
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagctgaatt cgccaccatg ggagggcacc cgcagctccg tctcgtcaag
      961 gcccttctcc ttctggggct gaaccccgtc tctgcctccc tccaggacca gcactgcgag
     1021 agcctgtccc tggccagcaa catctcagga ctgcagtgca acgcatccgt ggacctcatt
     1081 ggcacctgct ggccccgcag ccctgcgggg cagctagtgg ttcggccctg ccctgccttt
     1141 ttctatggtg tccgctacaa taccacaaac aatggctacc gggagtgcct ggccaatggc
     1201 agctgggccg cccgcgtgaa ttactccgag tgccaggaga tcctcaatga ggagaaaaaa
     1261 agcaaggtgc actaccatgt cgcagtcatc atcaactacc tgggccactg tatctccctg
     1321 gtggccctcc tggtggcctt tgtcctcttt ctgcggctca ggagcatccg gtgcctgcga
     1381 aacatcatcc actggaacct catctccgcc ttcatcctgc gcaacgccac ctggttcgtg
     1441 gtccagctaa ccatgagccc cgaggtccac cagagcaacg tgggctggtg caggttggtg
     1501 acagccgcct acaactactt ccatgtgacc aacttcttct ggatgttcgg cgagggctgc
     1561 tacctgcaca cagccatcgt gctcacctac tccactgacc ggctgcgcaa atggatgttc
     1621 atctgcattg gctggggtgt gcccttcccc atcattgtgg cctgggccat tgggaagctg
     1681 tactacgaca atgagaagtg ctggtttggc aaaaggcctg gggtgtacac cgactacatc
     1741 taccagggcc ccatgatcct ggtcctgctg atcaatttca tcttcctttt caacatcgtc
     1801 cgcatcctca tgaccaagct ccgggcatcc accacgtctg agaccattca gtacaggaag
     1861 gctgtgaaag ccactctggt gctgctgccc ctcctgggca tcacctacat gctgttcttc
     1921 gtcaatcccg gggaggatga ggtctcccgg gtcgtcttca tctacttcaa ctccttcctg
     1981 gaatccttcc agggcttctt tgtgtctgtg ttctactgtt tcctcaatag tgaggtccgt
     2041 tctgccatcc ggaagaggtg gcaccggtgg caggacaagc actcgatccg tgcccgagtg
     2101 gcccgtgcca tgtccatccc cacctcccca acccgtgtca gctttcacag catcaagcag
     2161 tccacagcag tcctcgagga ttacaaggat gacgacgata agggatccag cgcctggtcc
     2221 caccctcagt ttgagaaggg cggaggctct ggcggcggaa gcggaggatc tgcttggagc
     2281 cacccccagt tcgaaaagtg atgagggccc gtttaaaccc gctgatcagc ctcgactgtg
     2341 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     2401 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     2461 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     2521 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     2581 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2641 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2701 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2761 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2821 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2881 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     2941 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     3001 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
     3061 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3121 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3181 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     3241 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     3301 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     3361 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag
     3421 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     3481 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     3541 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     3601 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     3661 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     3721 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     3781 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     3841 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     3901 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     3961 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     4021 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     4081 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     4141 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     4201 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     4261 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     4321 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     4381 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     4441 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     4501 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     4561 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     4621 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     4681 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4741 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4801 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4861 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4921 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4981 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     5041 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5101 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5161 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5221 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5281 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5341 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5401 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     5461 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5521 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     5581 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     5641 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     5701 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     5761 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     5821 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     5881 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     5941 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     6001 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     6061 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     6121 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     6181 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     6241 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     6301 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     6361 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     6421 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     6481 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     6541 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     6601 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     6661 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     6721 agtgccacct gacgtc
//
