LOCUS       Exported                6864 bp DNA     circular SYN 24-JAN-2022
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 6864)
  AUTHORS   Jin Zhang Lab
  TITLE     Direct Submission
  JOURNAL   Exported Mar 14, 2022 from SnapGene 6.0.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6864
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
                     /note="color: #ffffff"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
                     /note="color: #ffffff; direction: RIGHT"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: RIGHT"
     regulatory      898..907
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
                     /regulatory_class="other"
     CDS             904..954
                     /codon_start=1
                     /label=LifeAct
                     /note="color: #800080"
                     /translation="MGVADLIKKFESISKEE"
     CDS             964..1635
                     /codon_start=1
                     /label=Dronpa (H193T)
                     /note="color: #008f00"
                     /translation="MSVIKPDMKIKLRMEGAVNGHPFAIEGVGLGKPFEGKQSMDLKVK
                     EGGPLPFAYDILTTVFCYGNRVFAKYPENIVDYFKQSFPEGYSWERSMNYEDGGICNAT
                     NDITLDGDCYIYEIRFDGVNFPANGPVMQKRTVKWEPSTEKLYVRDGVLKGDVNMALSL
                     EGGGHYRCDFKTTYKAKKVVQLPDYHFVDTHIEIKSHDKDYSNVNLHEHAEAHSELPRQ
                     AK"
     CDS             1660..2352
                     /codon_start=1
                     /label=TagRFP-T
                     /note="color: #ff2600"
                     /translation="MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTDMALK
                     LVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHK"
     promoter        complement(2416..2434)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
                     /note="color: #ffffff; direction: LEFT"
     polyA_signal    2460..2684
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
     rep_origin      2730..3158
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     promoter        3172..3502
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
                     /note="color: #ffffff; direction: RIGHT"
     rep_origin      3353..3488
                     /label=SV40 ori
                     /note="SV40 origin of replication"
                     /note="color: #ffff00"
     CDS             3569..4363
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /note="color: #ccffcc"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    4537..4658
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
                     /note="color: #a6acb3"
     primer_bind     complement(4707..4723)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     protein_bind    4731..4747
                     /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)."
                     /note="color: #31849b"
     promoter        complement(4755..4785)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This reverse directional feature has 3 segments:
                        1: 4755 .. 4761 / #ffffff / -10
                        2: 4762 .. 4779 / #ffffff
                        3: 4780 .. 4785 / #ffffff / -35"
     protein_bind    4800..4821
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
                     /note="color: #31849b"
     rep_origin      complement(5109..5697)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     CDS             complement(5868..6728)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 5868 .. 6659 / #ccffcc
                        2: 6660 .. 6728 / #ccffcc / signal sequence
                     Cleavage site after base 6659"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6729..6833)
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: LEFT"
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct 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 atgggtggac 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 gcttgccgcc
      901 accatgggcg tggccgacct gatcaagaag ttcgagagca tcagcaagga ggagggggat
      961 ccaatgagtg tgattaaacc agacatgaag atcaagctgc gtatggaagg cgctgtaaat
     1021 ggacacccgt tcgcgattga aggagttggc cttgggaagc ctttcgaggg aaaacagagt
     1081 atggacctta aagtcaaaga aggcggacct ctgcctttcg cctatgacat cttgacaact
     1141 gtgttctgtt acggcaacag ggtattcgcc aaatacccag aaaatatagt agactatttc
     1201 aagcagtcgt ttcctgaggg ctactcttgg gaacgaagca tgaattacga agacgggggc
     1261 atttgtaacg cgacaaacga cataaccctg gatggtgact gttatatcta tgaaattcga
     1321 tttgatggtg tgaactttcc tgccaatggt ccagttatgc agaagaggac tgtgaaatgg
     1381 gagccatcca ctgagaaatt gtatgtgcgt gatggagtgc tgaagggtga tgttaacatg
     1441 gctctgtcgc ttgaaggagg tggccattac cgatgtgact tcaaaactac ttataaagct
     1501 aagaaggttg tccagttgcc agactatcac tttgtggaca cacacattga gattaaaagc
     1561 cacgacaaag attacagtaa tgttaatctg catgagcatg ccgaagcgca ttctgagctg
     1621 ccgaggcagg ccaagagatc tgacgaggtg gacggtacca tgagcgagct gattaaggag
     1681 aacatgcaca tgaagctgta catggagggc accgtgaaca accaccactt caagtgcaca
     1741 tccgagggcg aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggtggtcgag
     1801 ggcggccctc tccccttcgc cttcgacatc ctggctacca gcttcatgta cggcagcaga
     1861 accttcatca accacaccca gggcatcccc gatttcttta agcagtcctt ccctgagggc
     1921 ttcacatggg agagagtcac cacatacgaa gacgggggcg tgctgaccgc tacccaggac
     1981 accagcctcc aggacggctg cctcatctac aacgtcaaga tcagaggggt gaacttccca
     2041 tccaacggcc ctgtgatgca gaagaaaaca ctcggctggg aggccaacac cgagatgctg
     2101 taccccgctg acggcggcct ggaaggcaga accgacatgg ccctgaagct cgtgggcggg
     2161 ggccacctga tctgcaactt caagaccaca tacagatcca agaaacccgc taagaacctc
     2221 aagatgcccg gcgtctacta tgtggaccac agactggaaa gaatcaagga ggccgacaaa
     2281 gagacctacg tcgagcagca cgaggtggct gtggccagat actgcgacct ccctagcaaa
     2341 ctggggcaca agtgagaatt ctgcagatat ccatcacact ggcggccgct cgagcatgca
     2401 tctagagggc cctattctat agtgtcacct aaatgctaga gctcgctgat cagcctcgac
     2461 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     2521 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     2581 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     2641 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     2701 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     2761 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     2821 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2881 tcggggcatc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     2941 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3001 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3061 ccctatctcg gtctattctt ttgatttata agggattttg gggatttcgg cctattggtt
     3121 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     3181 ttagggtgtg gaaagtcccc aggctcccca ggcaggcaga agtatgcaaa gcatgcatct
     3241 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3301 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     3361 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     3421 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
     3481 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga
     3541 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     3601 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     3661 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     3721 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
     3781 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     3841 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     3901 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     3961 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     4021 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     4081 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     4141 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     4201 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     4261 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     4321 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     4381 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     4441 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     4501 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     4561 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     4621 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     4681 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     4741 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     4801 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4861 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4921 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4981 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     5041 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     5101 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     5161 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     5221 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     5281 tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc
     5341 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     5401 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5461 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5521 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     5581 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     5641 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     5701 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     5761 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     5821 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     5881 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     5941 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     6001 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     6061 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     6121 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6181 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     6241 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     6301 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6361 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6421 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     6481 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6541 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     6601 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     6661 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     6721 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     6781 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     6841 ccccgaaaag tgccacctga cgtc
//
