LOCUS       Exported                9273 bp ds-DNA     circular SYN 23-JUL-2018
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9273)
  AUTHORS   Chen
  TITLE     Direct Submission
  JOURNAL   Exported Jul 24, 2018 from SnapGene Viewer 4.2.1
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9273
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        complement(1..105)
                     /gene="bla"
                     /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"
     misc_feature    874..936
                     /label=3' TR
     misc_feature    1113..1344
                     /label=Core Insulator
     misc_feature    1346..1366
                     /label=H1
     misc_feature    1367..1977
                     /label=SH LHA
     misc_feature    1886..1888
                     /label=PAM
     misc_feature    1889..1907
                     /label=SH gRNA 4
     misc_feature    1960..1968
                     /label=SHS231
     misc_feature    1978..2030
                     /label=Bxb1 attP
     promoter        2066..3244
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2297..3235
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     protein_bind    3256..3289
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             3298..4014
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             4021..4074
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             4078..5109
                     /codon_start=1
                     /label=HygromycinB Resistance
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     DLQ"
     misc_feature    5117..5158
                     /label=fC31 attP
     misc_feature    5159..5686
                     /label=SH RHA
     misc_feature    5337..5339
                     /label=PAM
     misc_feature    5340..5358
                     /label=SH gRNA 5
     misc_feature    5687..5707
                     /label=H2
     polyA_signal    5848..5965
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    complement(6222..6452)
                     /label=Core Insulator
     misc_feature    6727..6761
                     /label=5' TR
     primer_bind     complement(7252..7268)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    7276..7292
                     /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(7300..7330)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7345..7366
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(7654..8242)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(8413..9273)
                     /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"
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 caaggccaga ctagaggtac ccattgagtt
     1381 ctgaggtggt ttgtcataca gcaatggcaa tcagatatat ccacacaaat ataattttag
     1441 tttatatttc tgctactgca gttctcatct tattctgagg atatgtgaca aaataattct
     1501 ttcaaaaata ttgatgctgt gctagattac tattttgaat gaattactag acaaatactt
     1561 catatgtatc tttttttttt tttttttttt tttttttttt tttttttgag acggagtctc
     1621 gctctgtcgc ccaggctgga gtgcagtggc gggatctcgg ctcactgcaa tatgtatctt
     1681 attatgtggg tttacacatt atttatctta ttgatttaac ttcaaaacta aactttagtt
     1741 tagctcttgg gccctatctg gaaagggtca tcttttaatc accattaaat cactgaagtc
     1801 atcagtttat tcaaagtact ctgcacaaat tagcattctt tagtggttgt gaaataaata
     1861 gactttaaac ttatcattaa tattcccaat ggtactatgg gggaggcaaa attttctgtc
     1921 ttcttagtgg tttttttttt ttggctaggg ctaaggatta aactgtcaag cttcgaggtg
     1981 gtttgtctgg tcaaccaccg cggtctcagt ggtgtacggt acaaaccccg agtcgacctt
     2041 ctaggtcttg aaaggagtgg gaattggctc cggtgcccgt cagtgggcag agcgcacatc
     2101 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag
     2161 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg
     2221 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt
     2281 tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc tctttacggg
     2341 ttatggccct tgcgtgcctt gaattacttc cacctggctg cagtacgtga ttcttgatcc
     2401 cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag gagccccttc
     2461 gcctcgtgct tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg
     2521 gcaccttcgc gcctgtctcg ctgctttcga taagtctcta gccatttaaa atttttgatg
     2581 acctgctgcg acgctttttt tctggcaaga tagtcttgta aatgcgggcc aagatctgca
     2641 cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac
     2701 atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg ggtagtctca
     2761 agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc
     2821 ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc ttcccggccc
     2881 tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc
     2941 cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact ccacggagta
     3001 ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt tggagtacgt cgtctttagg
     3061 ttggggggag gggttttatg cgatggagtt tccccacact gagtgggtgg agactgaagt
     3121 taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg agtttggatc
     3181 ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat ttcaggtgtc
     3241 gtgaggaatt agcttataac ttcgtatagc atacattata cgaagttatc ctgcaggatg
     3301 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     3361 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     3421 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     3481 gtgaccacct tcacctacgg cgtgcagtgc ttcgcccgct accccgacca catgaagcag
     3541 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     3601 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     3661 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     3721 ctggagtaca actacaacag ccacaaggtc tatatcaccg ccgacaagca gaagaacggc
     3781 atcaaggtga acttcaagac ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     3841 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     3901 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     3961 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caaggctagc
     4021 gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg cccaaccatg
     4081 aaaaagcctg aactcaccgc tacctctgtc gagaagtttc tgatcgaaaa gttcgacagc
     4141 gtctccgacc tgatgcagct ctccgagggc gaagaatctc gggctttcag cttcgatgtg
     4201 ggagggcgtg gatatgtcct gcgggtgaat agctgcgccg atggtttcta caaagatcgc
     4261 tatgtttatc ggcactttgc atccgccgct ctccctattc ccgaagtgct tgacattggg
     4321 gagttcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt caccttgcaa
     4381 gacctgcctg aaaccgaact gcccgctgtt ctccagcccg tcgccgaggc catggatgcc
     4441 atcgctgccg ccgatcttag ccagaccagc gggttcggcc cattcggacc tcaaggaatc
     4501 ggtcaataca ctacatggcg cgatttcatc tgcgctattg ctgatcccca tgtgtatcac
     4561 tggcaaactg tgatggacga caccgtcagt gcctccgtcg cccaggctct cgatgagctg
     4621 atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgccga tttcggctcc
     4681 aacaatgtcc tgaccgacaa tggccgcata acagccgtca ttgactggag cgaggccatg
     4741 ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccctg gttggcttgt
     4801 atggagcagc agacccgcta cttcgagcgg aggcatcccg agcttgcagg atctcctcgg
     4861 ctccgggctt atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc
     4921 aatttcgatg atgcagcttg ggctcagggt cgctgcgacg caatcgtccg gtccggagcc
     4981 gggactgtcg ggcgtacaca aatcgcccgc agaagcgctg ccgtctggac cgatggctgt
     5041 gtggaagtgc tcgccgatag tggaaacaga cgccccagca ctcgtcctag ggcaaaggat
     5101 ctgcagtaat ctagaagtgc cccaactggg gtaacctttg agttctctca gttgggggac
     5161 tagtgcgtgg acaaagagca gtaaattgtg aaaatggaac aaggcaaggg ggcttagact
     5221 acagtagtta atcatcaaga agtgacaaaa aaaataatgg ttagttaata agatttgttt
     5281 aagcagattt ctcccagctt tagctctctg tctctggtga tcagaatgca ctccttcctt
     5341 cagactcagt gagcacatct tccacatgga agatttcttc tctagctttt aggaaatcca
     5401 gagaaccctt tttgtatctg ctgttttttt ttttttaaat gtctttaact caaaacaatt
     5461 tatgtgccag gatgacatat ctttggataa tgtgttctga actccttcag tacatacgta
     5521 tataaattaa agcaaatatt ttttatgata agctggcata atagtttcat aatttaatca
     5581 ctgatttaaa aatttaatta aaattatttt ttgatatttt gtgtaataat ttttgaggag
     5641 tatcttttgt gcttaatgag tggcagatga cacccatgtt cttagctacg cgttaactaa
     5701 gtaagcggcc gccccggtat cgacaatcaa cctctggatt acaaaatttg tgaaagattg
     5761 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     5821 ttgtatcatg cgttaactaa ctaaacttgt ttattgcagc ttataatggt tacaaataaa
     5881 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     5941 tgtccaaact catcaatgta tcttatcatg tctggaattg actcaaatga tgtcaattag
     6001 tctatcagaa gctatctggt ctcccttccg ggggacaaga catccctgtt taatatttaa
     6061 acagcagtgt tcccaaactg ggttcttata tcccttgctc tggtcaacca ggttgcaggg
     6121 tttcctgtcc tcacaggaac gaagtcccta aagaaacagt ggcagccagg tttagccccg
     6181 gaattgactg gattcctttt ttagggccca ttggtatggc tttttccccg tatcccccca
     6241 ggtgtctgca ggctcaaaga gcagcgagaa gcgttcagag gaaagcgatc ccgtgccacc
     6301 ttccccgtgc ccgggctgtc cccgcacgct gccggctcgg ggatgcgggg ggagcgccgg
     6361 accggagcgg agccccgggc ggctcgctgc tgccccctag cgggggaggg acgtaattac
     6421 atccctgggg gctttggggg ggggctgtcc ctgatatcta taacaagaaa atatatatat
     6481 aataagttat cacgtaagta gaacatgaaa taacaatata attatcgtat gagttaaatc
     6541 ttaaaagtca cgtaaaagat aatcatgcgt cattttgact cacgcggtcg ttatagttca
     6601 aaatcagtga cacttaccgc attgacaagc acgcctcacg ggagctccaa gcggcgactg
     6661 agatgtccta aatgcacagc gacggattcg cgctatttag aaagagagag caatatttca
     6721 agaatgcatg cgtcaatttt acgcagacta tctttctagg gttaatctag ctgcatcagg
     6781 atcatatcgt cgggtctttt ttccggctca gtcatcgccc aagctggcgc tatctgggca
     6841 tcggggagga agaagcccgt gccttttccc gcgaggttga agcggcatgg aaagagtttg
     6901 ccgaggatga ctgctgctgc attgacgttg agcgaaaacg cacgtttacc atgatgattc
     6961 gggaaggtgt ggccatgcac gcctttaacg gtgaactgtt cgttcaggcc acctgggata
     7021 ccagttcgtc gcggcttttc cggacacagt tccggatggt cagcccgaag cgcatcagca
     7081 acccgaacaa taccggcgac agccggaact gccgtgccgg tgtgcagatt aatgacagcg
     7141 gtgcggcgct gggatattac gtcagcgagg acgggtatcc tggctggatg ccgcagaaat
     7201 ggacatggat accccgtgag ttacccggcg ggcgcgcttg gcgtaatcat ggtcatagct
     7261 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     7321 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     7381 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     7441 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     7501 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     7561 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     7621 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     7681 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     7741 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     7801 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     7861 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     7921 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     7981 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     8041 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     8101 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     8161 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     8221 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     8281 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     8341 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     8401 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     8461 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     8521 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     8581 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     8641 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     8701 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     8761 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     8821 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     8881 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     8941 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     9001 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     9061 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     9121 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     9181 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     9241 aataagggcg acacggaaat gttgaatact cat
//
