LOCUS       sequence-61263-e        7446 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7446)
  AUTHORS   Nevozhay D, Zal T, Balazsi G
  TITLE     Transferring a synthetic gene circuit from yeast to mammalian cells.
  JOURNAL   Nat Commun. 2013 Feb 5;4:1451. doi: 10.1038/ncomms2471.
  PUBMED    23385595
REFERENCE   2  (bases 1 to 7446)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2013-02-5"; volume: "4:1451. doi"; pages: " 
            10.1038/ncomms2471"
COMMENT     Sequence Label: pDN-D2irTNG4kwh
FEATURES             Location/Qualifiers
     source          1..7446
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    814..832
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    840..858
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     intron          1041..1613
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     regulatory      1625..1634
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2297..2299,2300..2302,2303..3016)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2297..2299,2300..2302,2303..3013)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2342..2363)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2603..2622)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2950..2971
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3084..3104)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     primer_bind     complement(3457..3474)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3463..3687
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3733..4161
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3820..3839)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4030..4051
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4170..4190)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4175..4504
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4355..4490
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4417..4436
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4552..4599
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4618..4992
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    5122..5243
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5159..5178)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5213..5232
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5292..5308)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5292..5308)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5305..5327)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5316..5332
                     /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(join(5340..5346,5347..5364,5365..5370))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5385..5406
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5523..5540)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5694..6279)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5774..5793)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6450..7241,7242..7310))
                     /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"
     primer_bind     7073..7092
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7311..7415)
                     /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 gcatccctat cagtgataga gatcagatct
      841 ccctatcagt gatagagagc tgttttgacc tccatagaag acaccgggac cgatccagcc
      901 tccggactct agcgtttaaa cttaagcttg gtacccgggg atcctctagg gcctctgagc
      961 tattccagaa gtagtgaaga ggcttttttg gaggcctagg cttttgcaaa aagctccgga
     1021 tcgatcctga gaacttcagg gtgagtttgg ggacccttga ttgttctttc tttttcgcta
     1081 ttgtaaaatt catgttatat ggagggggca aagttttcag ggtgttgttt agaatgggaa
     1141 gatgtccctt gtatcaccat ggaccctcat gataattttg tttctttcac tttctactct
     1201 gttgacaacc attgtctcct cttattttct tttcattttc tgtaactttt tcgttaaact
     1261 ttagcttgca tttgtaacga atttttaaat tcacttttgt ttatttgtca gattgtaagt
     1321 actttctcta atcacttttt tttcaaggca atcagggtat attatattgt acttcagcac
     1381 agttttagag aacaattgtt ataattaaat gataaggtag aatatttctg catataaatt
     1441 ctggctggcg tggaaatatt cttattggta gaaacaacta catcctggtc atcatcctgc
     1501 ctttctcttt atggttacaa tgatatacac tgtttgagat gaggataaaa tactctgagt
     1561 ccaaaccggg cccctctgct aaccatgttc atgccttctt ctttttccta caggtcctgc
     1621 aggcgccacc atgggttcta gactggacaa gagcaaagtc ataaactctg ctctggaatt
     1681 actcaatgaa gtcggtatcg aaggcctgac gacaaggaaa ctcgctcaaa agctgggagt
     1741 tgagcagcct accctgtact ggcacgtgaa gaacaagcgg gccctgctcg atgccctggc
     1801 aatcgagatg ctggacaggc atcataccca cttctgcccc ctggaaggcg agtcatggca
     1861 agactttctg cggaacaacg ccaagtcatt ccgctgtgct ctcctctcac atcgcgacgg
     1921 ggctaaagtg catctcggca cccgcccaac agagaaacag tacgaaaccc tggaaaatca
     1981 gctcgcgttc ctgtgtcagc aaggcttctc cctggagaac gcactgtacg ctctgtccgc
     2041 cgtgggccac tttacactgg gctgcgtatt ggaggatcag gagcatcaag tagcaaaaga
     2101 ggaaagagag acacctacca ccgattctat gcccccactt ctgagacaag caattgagct
     2161 gttcgaccat cagggagccg aacctgcctt ccttttcggc ctggaactaa tcatatgtgg
     2221 cctggagaaa cagctaaagt gcgaaagcgg cgggccaaaa aagaagagaa agggtgacgg
     2281 tgctggttta attaacatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     2341 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     2401 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     2461 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     2521 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     2581 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     2641 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     2701 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     2761 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     2821 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     2881 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     2941 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     3001 cgagctgtac aagtaaaccg gtgcggccgc aatcaacctc tggattacaa aatttgtgaa
     3061 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     3121 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     3181 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     3241 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     3301 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     3361 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcc
     3421 tcgagtctag agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     3481 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     3541 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     3601 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     3661 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta
     3721 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3781 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3841 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3901 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     3961 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     4021 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     4081 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     4141 aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc
     4201 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     4261 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     4321 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     4381 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     4441 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     4501 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcagcacg tgttgacaat
     4561 taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg
     4621 gccaagttga ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag
     4681 ttctggaccg accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg
     4741 gtccgggacg acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac
     4801 accctggcct gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc
     4861 gtgtccacga acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg
     4921 tgggggcggg agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag
     4981 gagcaggact gacacgtgct acgagatttc gattccaccg ccgccttcta tgaaaggttg
     5041 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     5101 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     5161 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     5221 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     5281 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     5341 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     5401 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5461 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     5521 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5581 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5641 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5701 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5761 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5821 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5881 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5941 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     6001 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     6061 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     6121 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     6181 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tttttttgtt
     6241 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6301 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6361 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6421 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6481 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6541 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6601 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     6661 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6721 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     6781 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6841 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6901 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6961 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     7021 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     7081 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     7141 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     7201 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7261 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     7321 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7381 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     7441 gacgtc
//
