LOCUS       62056_8380        7974 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 7974)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7974
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        17..121
                     /label=AmpR promoter
     CDS             122..979
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      1153..1741
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    2029..2050
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        2065..2095
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2103..2119
                     /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     2127..2143
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2164..2182
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2219..2235
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2792..3689
                     /label=cmlc2
                     /note="Cardiac myosin light chain 2 promoter"
     CDS             3690..4406
                     /codon_start=1
                     /label=EGFP
                     /note="enhanced GFP"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    4529..4663
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_RNA        5062..5137
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     5150..5166
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5174..5297
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        5322..5352
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             5406..6083
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     CDS             6406..6708
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(6752..6876)
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(6895..6911)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
       61 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
      121 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
      181 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
      241 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
      301 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
      361 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
      421 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
      481 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
      541 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
      601 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
      661 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
      721 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
      781 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
      841 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
      901 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
      961 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     1021 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     1081 gaccaaaatg ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     1141 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     1201 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     1261 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     1321 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     1381 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     1441 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     1501 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     1561 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     1621 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     1681 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     1741 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     1801 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     1861 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     1921 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     1981 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     2041 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     2101 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
     2161 cgcaattaac cctcactaaa gggaacaaaa gctggagctc caccgcggtg gcggccgctc
     2221 tagaactagt ggatctgctg ggcttgctga aggtaggggg tcaagaacca gaggtgtaaa
     2281 gtacttgagt aattttactt gattactgta cttaagtatt atttttgggg atttttactt
     2341 tacttgagta caattaaaaa tcaatacttt tacttttact taattacatt tttttagaaa
     2401 aaaaagtact ttttactcct tacaatttta tttacagtca aaaagtactt attttttgga
     2461 gatcacttca ttctattttc ccttgctatt accaaaccaa ttgaattgcg ctgatgccca
     2521 gtttaattta aatgttattt attctgccta tgaaaatcgt tttcacatta tatgaaattg
     2581 gtcagacatg ttcattggtc ctttggaagt gacgtcatgt cacatctatt accacaatgc
     2641 acagcacctt gacctggaaa ttagggaaat tataacagtc aatcagtgga agaaaatgga
     2701 ggaagtatgt gattcatcag cagctgcgag cagcacagtc caaaatcagc cacaggatca
     2761 agagcacccg tggccgtatc ttcgcagatc taaagcttaa atcagttgtg ttaaataaga
     2821 gacattcaaa ataaatgtaa atgagctctc caaatcagca gacttaacat tctttaaaat
     2881 gattgattca atagtgataa aaatcaggca tagccagttg taactttaga taaattacag
     2941 aaaatgtcaa atacagagaa ccgattcttt tttatgatac atccaagcac acatttaaca
     3001 caatccaggc aaaccccgaa tttcacagtc acaagcactg tttgtacaag agctttgcct
     3061 aaggacacac agtctctata agtccaggtc gttggtttca ctcttatttt aaacatgtga
     3121 catttttcct gccatcctgt cttaggctgc tgtttgcttc attccatgtc acattaaatt
     3181 cctcagtagc accttttaca cacacagcca atcttttcca gaaaattcaa ttgctttgaa
     3241 gagataatgt gtgaacaaat ccatttagaa aaggaaaatt aagaatttgt aaaatcatct
     3301 gtaaattgtt ggcattcttc tgtatatgaa catcacatca tttacaggta aaggtctggt
     3361 cattaattat atgacaattt actggtatta ttttgtgaaa ggggctattt tcaatgcgtt
     3421 catccatcct tttcatccct caaatctctc attcacgtcc ccctccccat ctgcacactt
     3481 tatctcattt ccaccctgct ggaatctgag cacttgtgca gttatcaggg ctcctgtatt
     3541 taggaggctc tgggtgtcca tgtaggggac gaacagaaac actgcagacc tttatagaag
     3601 aacaattgat aagagtcctc atacataaag actccattag taagccagtg acccaggagc
     3661 ccagaccaac agcaaagcag acagtgacca tggtgagcaa gggcgaagag ctgttcaccg
     3721 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     3781 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     3841 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     3901 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     3961 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     4021 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     4081 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     4141 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     4201 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     4261 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     4321 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     4381 ctctcggcat ggacgagctg tacaagtaaa tctgatctag aggatcataa tcagccatac
     4441 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa
     4501 acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa
     4561 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     4621 tggtttgtcc aaactcatca atgtatctta tcatgtctgg atcatcatcg atgcgtcttt
     4681 tgttctggtc atcaaggagg ggggaatgtt ctgcgcatgc ctgtggggga gggagaagga
     4741 cacgtcactg aaaacgtccc tgcatcacac cgagacaccc aatcactcaa gccgagacca
     4801 gataattttg catatgcttt aaagtttgaa aaataccacg gtaaaccctc acacaaactc
     4861 tggatttgag atctttcagg ttttatcagt ttgcaggttt atgtcaccat gatatagggt
     4921 cagacttgat ctaagggagc tgaataagtg gtttagtcac tcaccacctc ccaaaaacat
     4981 acccagaagt ccctggtata tatagctctc cctccagctc ttggttcggc tagcactcct
     5041 cagtgacgag gagtcagtag cgttttagag ctagaaatag caagttaaaa taaggctagt
     5101 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttggtaccg taaaacgacg
     5161 gccagtgaat tatcaactat gtataataaa gttgaacgag aaacgtaaaa tgatataaat
     5221 atcaatatat taaattagat tttgcataaa aaacagacta cataatactg taaaacacaa
     5281 catatccagt cactatggcg gccgcattag gcaccccagg ctttacactt tatgcttccg
     5341 gctcgtataa tgtgtggatt ttgagttagg atccgtcgag attttcagga gctaaggaag
     5401 ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta
     5461 aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc
     5521 tggatattac ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct
     5581 ttattcacat tcttgcccgc ctgatgaatg ctcatccgga attccgtatg gcaatgaaag
     5641 acggtgagct ggtgatatgg gatagtgttc acccttgtta caccgttttc catgagcaaa
     5701 ctgaaacgtt ttcatcgctc tggagtgaat accacgacga tttccggcag tttctacaca
     5761 tatattcgca agatgtggcg tgttacggtg aaaacctggc ctatttccct aaagggttta
     5821 ttgagaatat gtttttcgtc tcagccaatc cctgggtgag tttcaccagt tttgatttaa
     5881 acgtggccaa tatggacaac ttcttcgccc ccgttttcac catgggcaaa tattatacgc
     5941 aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt tgtgatggct
     6001 tccatgtcgg cagaatgctt aatgaattac aacagtactg cgatgagtgg caggcggggc
     6061 gtaatctaga ggatccggct tactaaaagc cagataacag tatgcgtatt tgcgcgctga
     6121 tttttgcggt ataagaatat atactgatat gtatacccga agtatgtcaa aaagaggtat
     6181 gctatgaagc agcgtattac agtgacagtt gacagcgaca gctatcagtt gctcaaggca
     6241 tatatgatgt caatatctcc ggttcggtaa gcacaaccat gcagaatgaa gcccgtcgtc
     6301 tgcgtgccga acgctggaaa gcggaaaatc aggaagggat ggctgaggtc gcccggttta
     6361 ttgaaatgaa cggctccttt gccgacgaga acaggggctg gtgaaatgca gtttaaggtt
     6421 tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt
     6481 gacacgcccg ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa
     6541 gtcccccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc
     6601 accgatatgg ccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac
     6661 cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc
     6721 tcccttatac acagccagtc tgcaggtcga ccatagtgac tggatatgtt gtgttttaca
     6781 gtattatgta gtctgttttt tatgcaaaat ctaatttaat atattgatat ttatatcatt
     6841 ttacgtttct cgttcaactt ttctatacaa agttgatagc ttggcgtaat catggtcata
     6901 gctgtttcct gctcgagggc ccatctggcc tgtgtttcag acaccaggga gtctctgctc
     6961 acgtttcctg ctatttgcag cctctctatc aagactaata cacctcttcc cgcatcggct
     7021 gcctgtgaga ggcttttcag cactgcagga ttgcttttca gccccaaaag agctaggctt
     7081 gacactaaca attttgagaa tcagcttcta ctgaagttaa atctgaggtt ttacaacttt
     7141 gagtagcgtg tactggcatt agattgtctg tcttatagtt tgataattaa atacaaacag
     7201 ttctaaagca ggataaaacc ttgtatgcat ttcatttaat gttttttgag attaaaagct
     7261 taaacaagaa tctctagttt tctttcttgc ttttactttt acttccttaa tactcaagta
     7321 caattttaat ggagtacttt tttactttta ctcaagtaag attctagcca gatactttta
     7381 cttttaattg agtaaaattt tccctaagta cttgtacttt cacttgagta aaatttttga
     7441 gtacttttta cacctctgtc aagaactcct ggacaaacct ctgacctgtg tggaacagag
     7501 tggatatggg tgtctgaaca gatattcacg tcttttgcag atcagagggc atttctggtg
     7561 ttgtgcaatg acctgggtcc aacaggtagg cgtactcgta tccaagagca ttgttggaca
     7621 ggaggtagtc tccattccta taaagaggaa ggaatggcac catgtagtag ccgtcattgt
     7681 ggccaattgg tgcattggta cgtgggtaga tggaccgggg aggctgatga gttcttagcc
     7741 agcgtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     7801 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     7861 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     7921 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcac
//
