LOCUS       Exported                6038 bp ds-DNA     circular SYN 21-NOV-2019
DEFINITION  synthetic circular DNA
ACCESSION   VB191115-1272dwu
VERSION     .
KEYWORDS    VB191115-1272dwu.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6038)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported to SnapGene on Nov 15, 2019
REFERENCE   2  (bases 1 to 6038)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Feb 21, 2025 from SnapGene 4.2.11
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6038
                     /organism="synthetic DNA construct"
     protein_bind    1..21
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        22..1754
                     /label=CAG
                     /note="/application_notes=Strong promoter."
                     /note="/description=CMV early enhancer fused to modified 
                     chicken beta-actin promoter"
                     /note="/full_name=Cytomegalovirus early enhancer fused with
                     chicken beta-actin promoter"
                     /note="/official_designation=CAG"
                     /note="/uuid=420ea94873db443a902370384d17a441"
     enhancer        25..404
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        406..683
                     /label=chicken beta-actin promoter
     intron          684..1700
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     protein_bind    1754..1778
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    1779..1784
                     /label=Kozak
                     /label=nonstandard type: Miscellaneous
                     /note="/application_notes=Facilitates translation 
                     initiation of ATG start codon downstream of the Kozak 
                     sequence."
                     /note="/description=Kozak translation initiation sequence"
                     /note="/full_name=Kozak"
                     /note="/official_designation=Kozak"
                     /note="/uuid=5f18b25a1234443494a618363f09f052"
     misc_feature    1785..2534
                     /label={NLS-mScarletI-NLS}
                     /label=nonstandard type: ORF
                     /note="/application_notes="
                     /note="/official_designation={NLS-mScarletI-NLS}"
                     /note="/uuid=94001b55013a4915b7cc30e3277f83f7"
     CDS             1788..1808
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1809..2504
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     CDS             2505..2531
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     protein_bind    complement(2535..2559)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2559..3146
                     /label=IRES
                     /label=nonstandard type: Linker
                     /note="/application_notes=Recruits ribosome to initiate 
                     translation internally on a transcript independent of its 
                     5' end. Multiple proteins can be made from a polycistronic 
                     transcript containing multiple ORFs separated by IRES."
                     /note="/description=Encephalomyocarditis virus internal 
                     ribosome entry site"
                     /note="/full_name=Encephalomyocarditis virus internal 
                     ribosome entry site"
                     /note="/official_designation=IRES"
                     /note="/uuid=344233c15e2a4a5a9566583bfc1af3cb"
     misc_feature    2560..3146
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3147..3746
                     /label=Puro
                     /label=nonstandard type: ORF
                     /note="/application_notes=Allows cells to be resistant to 
                     puromycin."
                     /note="/description=Puromycin resistance gene"
                     /note="/full_name=Puromycin resistance gene"
                     /note="/official_designation=Puro"
                     /note="/uuid=39d7413208494d9291d10b21b13ca44c"
     CDS             3147..3746
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     protein_bind    complement(3747..3767)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    3786..4007
                     /label=SV40 late pA
                     /note="/application_notes=Allows transcription termination 
                     and polyadenylation of mRNA transcribed by Pol II RNA 
                     polymerase."
                     /note="/description=Simian virus 40 late polyadenylation 
                     signal"
                     /note="/official_designation=SV40 late pA"
                     /note="/uuid=d23bf533e0924dfcb932fc3ddd77e791"
     polyA_signal    3795..3916
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    4007..4008
                     /label=No marker
                     /label=nonstandard type: Marker
                     /note="/application_notes="
                     /note="/official_designation=No marker(ns)"
                     /note="/uuid=72e1edd2c1a540b28efbb477521f8696"
     rep_origin      complement(4203..4791)
                     /direction=LEFT
                     /label=pUC ori
                     /note="/application_notes=Facilitates plasmid replication 
                     in E. coli; regulates high-copy plasmid number (500-700)."
                     /note="/description=pUC origin of replication"
                     /note="/full_name=pUC origin of replication"
                     /note="/official_designation=pUC ori"
                     /note="/uuid=41c627938a364ebfa136b55444385c5b"
     misc_feature    complement(4962..5822)
                     /label=Ampicillin
                     /label=nonstandard type: ORF
                     /note="/application_notes=Allows E. coli to be resistant to
                     ampicillin."
                     /note="/description=Ampicillin resistance gene"
                     /note="/full_name=Ampicillin resistance gene"
                     /note="/official_designation=Ampicillin"
                     /note="/uuid=0bc7d58885794858b29e98fe171ff1d5"
     CDS             complement(4962..5822)
                     /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(5823..5927)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 caactttgta tagaaaagtt gctcgacatt gattattgac tagttattaa tagtaatcaa
       61 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      121 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      181 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      241 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
      301 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      361 ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca
      421 cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta
      481 ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccaggcg
      541 gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc
      601 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata
      661 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct
      721 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
      781 gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggcttg
      841 tttcttttct gtggctgcgt gaaagccttg aggggctccg ggagggccct ttgtgcgggg
      901 ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg
      961 cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg
     1021 tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg cggggggggc tgcgagggga
     1081 acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg
     1141 tcgggctgca accccccctg cacccccctc cccgagttgc tgagcacggc ccggcttcgg
     1201 gtgcggggct ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc
     1261 aggtgggggt gccgggcggg gcggggccgc ctcgggccgg ggagggctcg ggggaggggc
     1321 gcggcggccc ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc cattgccttt
     1381 tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt gcggagccga
     1441 aatctgggag gcgccgccgc accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg
     1501 gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc
     1561 ctctccagcc tcggggctgt ccgcgggggg acggctgcct tcggggggga cggggcaggg
     1621 cggggttcgg cttctggcgt gtgaccggcg gctctagagc ctctgctaac catgttcatg
     1681 ccttcttctt tttcctacag ctcctgggca acgtgctggt tattgtgctg tctcatcatt
     1741 ttggcaaaga attgcaagtt tgtacaaaaa agcaggctgc caccatgcca aaaaagaaga
     1801 gaaaggtaat ggtgagcaag ggcgaggcag tgatcaagga gttcatgcgg ttcaaggtgc
     1861 acatggaggg ctccatgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
     1921 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttct
     1981 cctgggacat cctgtcccct cagttcatgt acggctccag ggccttcatc aagcaccccg
     2041 ccgacatccc cgactactat aagcagtcct tccccgaggg cttcaagtgg gagcgcgtga
     2101 tgaacttcga ggacggcggc gccgtgaccg tgacccagga cacctccctg gaggacggca
     2161 ccctgatcta caaggtgaag ctccgcggca ccaacttccc tcctgacggc cccgtaatgc
     2221 agaagaagac aatgggctgg gaagcgtcca ccgagcggtt gtaccccgag gacggcgtgc
     2281 tgaagggcga cattaagatg gccctgcgcc tgaaggacgg cggccgctac ctggcggact
     2341 tcaagaccac ctacaaggcc aagaagcccg tgcagatgcc cggcgcctac aacgtcgacc
     2401 gcaagttgga catcacctcc cacaacgagg actacaccgt ggtggaacag tacgaacgct
     2461 ccgagggccg ccactccacc ggcggcatgg acgagctgta caagcctgct gccaagaggg
     2521 tcaagttgga ctagacccag ctttcttgta caaagtgggc ccctctccct cccccccccc
     2581 taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt
     2641 ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt
     2701 gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt
     2761 cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct
     2821 ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt
     2881 ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt
     2941 ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa
     3001 ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta
     3061 gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa
     3121 aacacgatga taatatggcc acaaccatga ccgagtacaa gcccacggtg cgcctcgcca
     3181 cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
     3241 ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac
     3301 tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg
     3361 cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg
     3421 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc
     3481 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc
     3541 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg
     3601 agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg
     3661 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt
     3721 gcatgacccg caagcccggt gcctgacaac tttattatac atagttgatg gccggccgct
     3781 tcgagcagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg
     3841 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     3901 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     3961 ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtagcg gccgcggcgc
     4021 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     4081 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     4141 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4201 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4261 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4321 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct ctcttcggga
     4381 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4441 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4501 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4561 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4621 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     4681 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4741 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4801 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4861 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     4921 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4981 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     5041 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     5101 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     5161 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     5221 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     5281 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     5341 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     5401 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     5461 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     5521 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     5581 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     5641 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     5701 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     5761 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     5821 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     5881 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     5941 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     6001 cgtatcacga ggccctttcg tcggcgcgcc gcggccgc
//
