LOCUS       Exported                7227 bp DNA     circular SYN 22-JUN-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 7227)
  AUTHORS   Sohum Mehta
  TITLE     Direct Submission
  JOURNAL   Exported Jun 22, 2021 from SnapGene 5.3.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7227
                     /organism="recombinant plasmid"
                     /lab_host="Mammalian Cells"
                     /mol_type="other DNA"
     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"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             922..939
                     /codon_start=1
                     /label=6His
                     /translation="HHHHHH"
     CDS             943..975
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             979..1002
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             1009..1407
                     /codon_start=1
                     /product="dimeric small ultra-red fluorescent protein 
                     derived from a cyanobacterial phycobiliprotein (Rodriguez 
                     et al., 2016)"
                     /label=smURFP
                     /translation="MAKTSEQRVNIATLLTENKKKIVDKASQDLWRRHPDLIAPGGIAF
                     SQRDRALCLRDYGWFLHLITFCLLAGDKGPIESIGLISIREMYNSLGVPVPAMMESIRC
                     LKEASLSLLDEEDANETAPYFDYIIKAMS"
     CDS             1432..1593
                     /codon_start=1
                     /label=WW Domain
                     /translation="MADEEKLPPGWEKRMSRSSGRVYYFNHITNASQWERPSGNSSSGG
                     KNGQGEPAR"
     CDS             1600..1947
                     /codon_start=1
                     /label=EV Linker
                     /translation="SAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGS
                     AGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGG
                     SAGGSAGGSAGG"
     CDS             1954..2007
                     /codon_start=1
                     /label=ERKsub_DD
                     /translation="PDVPRTPVDKAKLSFQFP"
     CDS             2023..2715
                     /codon_start=1
                     /label=StagRFP
                     /translation="MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTVMALK
                     LVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHK"
     promoter        complement(2779..2797)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     polyA_signal    2823..3047
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3093..3521
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        3535..3865
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3716..3851
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3932..4726
                     /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))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    4900..5021
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5070..5086)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5094..5110
                     /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(5118..5148)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5163..5184
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5472..6060)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6231..7091)
                     /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"
     promoter        complement(7092..7196)
                     /gene="bla"
                     /label=AmpR promoter
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 gcttgcggcc
      901 gccaccatgg tgcggggttc tcatcatcat catcatcatg gtatggctag catgactggt
      961 ggacagcaaa tgggtcggga tctgtacgac gatgacgata aggatcccat ggctaagact
     1021 tccgaacaga gggtgaacat tgctacactg ctgacagaaa ataagaagaa aatcgtggat
     1081 aaggcttccc aggatctgtg gcggagacac ccagacctga tcgcaccagg aggaattgct
     1141 ttctctcaga gggaccgcgc tctgtgcctg cgagattacg gctggttcct gcatctgatc
     1201 accttttgtc tgctggccgg agataagggc cccatcgagt ctattgggct gatcagtatt
     1261 cgagaaatgt ataactcact gggagtgccc gtccctgcaa tgatggagag cattagatgc
     1321 ctgaaagaag ccagcctgtc cctgctggac gaagaggacg ccaacgagac cgcaccctac
     1381 tttgattaca ttattaaggc tatgagcgac gagctgtaca agcgcatgca catggcggac
     1441 gaggagaagc tgccgcccgg ctgggagaag cgcatgagcc gcagctcagg ccgagtgtac
     1501 tacttcaacc acatcactaa cgccagccag tgggagcggc ccagcggcaa cagcagcagt
     1561 ggtggcaaaa acgggcaggg ggagcctgcc aggggtacca gtgctggtgg tagtgctggt
     1621 ggtagtgctg gtggtagtgc tggtggtagt gctggtggtt ccggcagtgc tggtggtagt
     1681 gctggtggta gtaccagtgc tggtggtagt gctggtggta gtgctggtgg tagtgctggt
     1741 ggtagtgctg gtggttccgg cagtgctggt ggtagtgctg gtggtagtac cagtgctggt
     1801 ggtagtgctg gtggtagtgc tggtggtagt gctggtggta gtgctggtgg ttccggcagt
     1861 gctggtggta gtgctggtgg tagtaccagt gctggtggta gtgctggtgg tagtgctggt
     1921 ggtagtgctg gtggtagtgc tggtggttcc ggaccagatg tccctagaac tccagtggat
     1981 aaagcaaagc tgtcattcca attcccccgc gcacgggagc tcatgagcga gctgattaag
     2041 gagaacatgc acatgaagct gtacatggag ggcaccgtga acaaccacca cttcaagtgc
     2101 acatccgagg gcgaaggcaa gccctacgag ggcacccaga ccatgagaat caaggtggtc
     2161 gagggcggcc ctctcccctt cgccttcgac atcctggcta ccagcttcat gtacggcagc
     2221 agaaccttca tcaaccacac ccagggcatc cccgatttct ttaagcagtc cttccctgag
     2281 ggcttcacat gggagagagt caccacatac gaagacgggg gcgtgctgac cgctacccag
     2341 gacaccagcc tccaggacgg ctgcctcatc tacaacgtca agatcagagg ggtgaacttc
     2401 ccatccaacg gccctgtgat gcagaagaaa acactcggct gggaggccaa caccgagatg
     2461 ctgtaccccg ctgacggcgg cctggaaggc agaaccgtca tggccctgaa gctcgtgggc
     2521 gggggccacc tgatctgcaa cttcaagacc acatacagat ccaagaaacc cgctaagaac
     2581 ctcaagatgc ccggcgtcta ctatgtggac cacagactgg aaagaatcaa ggaggccgac
     2641 aaagagacct acgtcgagca gcacgaggtg gctgtggcca gatactgcga cctccctagc
     2701 aaactggggc acaagtaaga attctgcaga tatccatcac actggcggcc gctcgagcat
     2761 gcatctagag ggccctattc tatagtgtca cctaaatgct agagctcgct gatcagcctc
     2821 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     2881 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     2941 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     3001 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     3061 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     3121 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     3181 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     3241 aaatcggggc atccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     3301 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     3361 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     3421 caaccctatc tcggtctatt cttttgattt ataagggatt ttggggattt cggcctattg
     3481 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     3541 cagttagggt gtggaaagtc cccaggctcc ccaggcaggc agaagtatgc aaagcatgca
     3601 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3661 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     3721 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     3781 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
     3841 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc
     3901 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     3961 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     4021 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     4081 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     4141 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     4201 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     4261 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     4321 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     4381 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     4441 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     4501 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     4561 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     4621 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     4681 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     4741 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     4801 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     4861 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     4921 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     4981 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     5041 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     5101 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     5161 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     5221 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5281 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5341 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5401 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5461 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5521 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5581 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5641 ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag
     5701 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5761 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5821 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5881 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     5941 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     6001 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     6061 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     6121 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     6181 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6241 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6301 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6361 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6421 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6481 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6541 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6601 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6661 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6721 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6781 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6841 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6901 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6961 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     7021 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     7081 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7141 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     7201 tttccccgaa aagtgccacc tgacgtc
//
