LOCUS       Exported                8694 bp ds-DNA     circular SYN 15-JUL-2023
DEFINITION  Neelamegham_R5S_1_EGFP-P2A-dTomato-PS9_C1     coverage: 1.23e+03x.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8694)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Dec 9, 2024 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8694
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        185..564
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        565..768
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        813..831
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      892..901
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             898..1614
                     /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             898..1614
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             1630..1686
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1693..2394
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLYGMDELYK"
     misc_feature    2433..3524
                     /label=SARS-CoV-2 PS9
     misc_feature    3578..4166
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             complement(4049..4060)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4241..4465
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4511..4939
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        4953..5282
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5133..5268
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5349..6143
                     /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    6317..6438
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6487..6503)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6511..6527
                     /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(6535..6565)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6580..6601
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(6889..7477)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7648..8508)
                     /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(8509..8613)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
       61 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
      121 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
      181 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
      241 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      301 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      361 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      421 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      481 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      541 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
      601 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      661 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      721 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
      781 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
      841 gctggctagc ttgttagacg aagcttgggc tgcaggtccg atccaccggt cgccaccatg
      901 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
      961 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     1021 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1081 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     1141 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     1201 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     1261 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     1321 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     1381 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     1441 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     1501 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     1561 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagggaagc
     1621 ggcagcggcg ccaccaactt cagcctgctg aagcaggccg gcgacgtgga ggagaacccc
     1681 ggccccgtta acatggtgag caagggcgag gaggtcatca aagagttcat gcgcttcaag
     1741 gtgcgcatgg agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc
     1801 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc
     1861 ttcgcctggg acatcctgtc cccccagttc atgtacggct ccaaggcgta cgtgaagcac
     1921 cccgccgaca tccccgatta caagaagctg tccttccccg agggcttcaa gtgggagcgc
     1981 gtgatgaact tcgaggacgg cggtctggtg accgtgaccc aggactcctc cctgcaggac
     2041 ggcacgctga tctacaaggt gaagatgcgc ggcaccaact tcccccccga cggccccgta
     2101 atgcagaaga agaccatggg ctgggaggcc tccaccgagc gcctgtaccc ccgcgacggc
     2161 gtgctgaagg gcgagatcca ccaggccctg aagctgaagg acggcggcca ctacctggtg
     2221 gagttcaaga ccatctacat ggccaagaag cccgtgcaac tgcccggcta ctactacgtg
     2281 gacaccaagc tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgag
     2341 cgctccgagg gccgccacca cctgttcctg tacggcatgg acgagctgta caagtaaatg
     2401 cggccgcctc ctttaagaag gagatataga tctctgtagg tcccaaacaa gctagtctta
     2461 atggagtcac attaattgga gaagccgtaa aaacacagtt caattattat aagaaagttg
     2521 atggtgttgt ccaacaatta cctgaaactt actttactca gagtagaaat ttacaagaat
     2581 ttaaacccag gagtcaaatg gaaattgatt tcttagaatt agctatggat gaattcattg
     2641 aacggtataa attagaaggc tatgccttcg aacatatcgt ttatggagat tttagtcata
     2701 gtcagttagg tggtttacat ctactgattg gactagctaa acgttttaag gaatcacctt
     2761 ttgaattaga agattttatt cctatggaca gtacagttaa aaactatttc ataacagatg
     2821 cgcaaacagg ttcatctaag tgtgtgtgtt ctgttattga tttattactt gatgattttg
     2881 ttgaaataat aaaatcccaa gatttatctg tagtttctaa ggttgtcaaa gtgactattg
     2941 actatacaga aatttcattt atgctttggt gtaaagatgg ccatgtagaa acattttacc
     3001 caaaattaca atctagtcaa gcgtggcaac cgggtgttgc tatgcctaat ctttacaaaa
     3061 tgcaaagaat gctattagaa aagtgtgacc ttcaaaatta tggtgatagt gcaacattac
     3121 ctaaaggcat aatgatgaat gtcgcaaaat atactcaact gtgtcaatat ttaaacacat
     3181 taacattagc tgtaccctat aatatgagag ttatacattt tggtgctggt tctgataaag
     3241 gagttgcacc aggtacagct gttttaagac agtggttgcc tacgggtacg ctgcttgtcg
     3301 attcagatct taatgacttt gtctctgatg cagattcaac tttgattggt gattgtgcaa
     3361 ctgtacatac agctaataaa tgggatctca ttattagtga tatgtacgac cctaagacta
     3421 aaaatgttac aaaagaaaat gactctaaag agggtttttt cacttacatt tgtgggttta
     3481 tacaacaaaa gctagctctt ggaggttccg tggctataaa gatagatccc aactccataa
     3541 gtgtgtcgac atataaggat ccactcgacg actcgacaat caacctctgg attacaaaat
     3601 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     3661 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     3721 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     3781 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     3841 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     3901 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     3961 gttgtcgggg aagctgacgt cctttccatg gctgctcgcc tgtgttgcca cctggattct
     4021 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     4081 cggcctgctg ccggctctgc ggcctcttcc gcatcttcgc cttcgccctc agacgagtcg
     4141 gatctccctt tgggccgcct ccccgcctgg aattaattcg agctcggtac cttgattctc
     4201 gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     4261 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     4321 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     4381 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     4441 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     4501 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     4561 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     4621 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     4681 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     4741 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     4801 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     4861 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     4921 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     4981 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     5041 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     5101 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     5161 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     5221 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     5281 aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat
     5341 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     5401 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     5461 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     5521 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     5581 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     5641 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     5701 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     5761 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     5821 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     5881 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     5941 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     6001 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     6061 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     6121 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     6181 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     6241 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     6301 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     6361 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     6421 actcatcaat gtatcttatc atgtctgtat accctccacc tctagctaga gcttggcgta
     6481 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     6541 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     6601 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     6661 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     6721 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     6781 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     6841 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     6901 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     6961 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     7021 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     7081 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     7141 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     7201 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     7261 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     7321 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     7381 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     7441 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     7501 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     7561 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     7621 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     7681 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     7741 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     7801 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     7861 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     7921 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     7981 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     8041 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     8101 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     8161 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     8221 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     8281 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     8341 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     8401 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     8461 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     8521 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     8581 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     8641 cctccacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatc
//
