LOCUS       Exported                6780 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pDN-D2irG4kwh
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6780)
  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 6780)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6780
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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"
     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
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1631..2350
                     /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             1631..2347
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1676..1697)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1937..1956)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2284..2305
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2418..2438)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     primer_bind     complement(2791..2808)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2797..3021
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3067..3495
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3154..3173)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3364..3385
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3504..3524)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3509..3838
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3689..3824
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3751..3770
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3886..3933
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3952..4326
                     /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    4456..4577
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4493..4512)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4547..4566
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4626..4642)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4626..4642)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4639..4661)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4650..4666
                     /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(4674..4704)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4719..4740
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4857..4874)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5028..5613)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5108..5127)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5784..6644)
                     /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     6407..6426
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6645..6749)
                     /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 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     1681 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     1741 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     1801 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     1861 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     1921 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     1981 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     2041 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     2101 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     2161 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     2221 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     2281 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     2341 gtacaagtaa accggtgcgg ccgcaatcaa cctctggatt acaaaatttg tgaaagattg
     2401 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     2461 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     2521 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     2581 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     2641 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     2701 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcctcgagt
     2761 ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
     2821 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     2881 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     2941 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     3001 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     3061 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3121 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     3181 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3241 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     3301 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3361 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     3421 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     3481 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     3541 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     3601 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3661 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     3721 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     3781 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     3841 gctcccggga gcttgtatat ccattttcgg atctgatcag cacgtgttga caattaatca
     3901 tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac catggccaag
     3961 ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg
     4021 accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg
     4081 gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg
     4141 gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc
     4201 acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg
     4261 cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag
     4321 gactgacacg tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     4381 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     4441 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     4501 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     4561 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     4621 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     4681 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     4741 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4801 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     4861 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4921 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4981 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     5041 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     5101 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     5161 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     5221 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     5281 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     5341 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     5401 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     5461 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     5521 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtttttt tgtttgcaag
     5581 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     5641 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5701 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5761 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5821 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5881 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5941 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     6001 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     6061 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     6121 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     6181 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     6241 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     6301 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     6361 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     6421 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     6481 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     6541 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     6601 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6661 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6721 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
