LOCUS       Exported                6761 bp ds-DNA     circular SYN 21-JUN-2023
DEFINITION  synthetic circular DNA
ACCESSION   VB191112-1110amt
VERSION     .
KEYWORDS    VB191112-1110amt.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6761)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported to SnapGene on Nov 13, 2019
REFERENCE   2  (bases 1 to 6761)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Feb 21, 2025 from SnapGene 4.2.11
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6761
                     /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"
     CDS             1785..3257
                     /codon_start=1
                     /label=hNEUROG3[NM_020999.3]*
                     /label=nonstandard type: ORF
                     /note="/application_notes="
                     /note="/official_designation=hNEUROG3[NM_020999.3]"
                     /note="/uuid=18915fce4b1949ed90b06b7b5b2064ee"
                     /translation="MTPQPSGAPTVQVTRETERSFPRASEDEVTCPTSAPPSPTRTRGN
                     CAEAEEGGCRGAPRKLRARRGGRSRPKSELALSKQRRSRRKKANDRERNRMHNLNSALD
                     ALRGVLPTFPDDAKLTKIETLRFAHNYIWALTQTLRIADHSLYALEPPAPHCGELGSPG
                     GSPGDWGSLYSPVSQAGSLSPAASLEERPGLLGATFSACLSPGSLAFSDFLGSAGSAAG
                     SGEFDYKDHDGDYKDHDIDYKDDDDKDINTPGINLIKEDMRVKVHMEGNVNGHAFVIEG
                     EGKGKPYEGTQTANLTVKEGAPLPFSYDILTTAVHYGNRVFTKYPEDIPDYFKQSFPEG
                     YSWERTMTFEDKGICTIRSDISLEGDCFFQNVRFKGTNFPPNGPVMQKKTLKWEPSTEK
                     LHVRDGLLVGNINMALLLEGGGHYLCDFKTTYKAKKVVQLPDAHFVDHRIEILGNDSDY
                     NKVKLYEHAVARYSPLPSQVWDIYPYDVPDYA"
     CDS             1785..2426
                     /codon_start=1
                     /label=Ngn3
                     /translation="MTPQPSGAPTVQVTRETERSFPRASEDEVTCPTSAPPSPTRTRGN
                     CAEAEEGGCRGAPRKLRARRGGRSRPKSELALSKQRRSRRKKANDRERNRMHNLNSALD
                     ALRGVLPTFPDDAKLTKIETLRFAHNYIWALTQTLRIADHSLYALEPPAPHCGELGSPG
                     GSPGDWGSLYSPVSQAGSLSPAASLEERPGLLGATFSACLSPGSLAFSDFL"
     CDS             2427..2462
                     /codon_start=1
                     /product="From WALDO ET AL 
                     http://www.nature.com/nbt/journal/v17/n7/full/nbt0799_691.h
                     tml"
                     /label=Linker
                     /translation="GSAGSAAGSGEF"
     CDS             2463..2528
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2535..3221
                     /codon_start=1
                     /product="monomeric photoswitchable green-to-red 
                     fluorescent protein from Dendronephthya"
                     /label=Dendra2
                     /note="mammalian codon-optimized"
                     /translation="NTPGINLIKEDMRVKVHMEGNVNGHAFVIEGEGKGKPYEGTQTAN
                     LTVKEGAPLPFSYDILTTAVHYGNRVFTKYPEDIPDYFKQSFPEGYSWERTMTFEDKGI
                     CTIRSDISLEGDCFFQNVRFKGTNFPPNGPVMQKKTLKWEPSTEKLHVRDGLLVGNINM
                     ALLLEGGGHYLCDFKTTYKAKKVVQLPDAHFVDHRIEILGNDSDYNKVKLYEHAVARYS
                     PLPSQVW"
     CDS             3228..3254
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    3228..3254
                     /label=HA tag
                     /label=insertion_seq
                     /note="vntifkey=14"
     protein_bind    complement(3258..3282)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    3282..3869
                     /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    3283..3869
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3870..4469
                     /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             3870..4469
                     /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(4470..4490)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    4509..4730
                     /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    4518..4639
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    4730..4731
                     /label=No marker
                     /label=nonstandard type: ORF
                     /note="/application_notes="
                     /note="/official_designation=No marker(ns)"
                     /note="/uuid=72e1edd2c1a540b28efbb477521f8696"
     rep_origin      complement(4926..5514)
                     /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(5685..6545)
                     /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(5685..6545)
                     /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(6546..6650)
                     /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 caccatgacg cctcaaccct
     1801 cgggtgcgcc cactgtccaa gtgacccgtg agacggagcg gtccttcccc agagcctcgg
     1861 aagacgaagt gacctgcccc acgtccgccc cgcccagccc cactcgcaca cgggggaact
     1921 gcgcagaggc ggaagaggga ggctgccgag gggccccgag gaagctccgg gcacggcgcg
     1981 ggggacgcag ccggcctaag agcgagttgg cactgagcaa gcagcgacgg agtcggcgaa
     2041 agaaggccaa cgaccgcgag cgcaatcgaa tgcacaacct caactcggca ctggacgccc
     2101 tgcgcggtgt cctgcccacc ttcccagacg acgcgaagct caccaagatc gagacgctgc
     2161 gcttcgccca caactacatc tgggcgctga ctcaaacgct gcgcatagcg gaccacagct
     2221 tgtacgcgct ggagccgccg gcgccgcact gcggggagct gggcagccca ggcggttccc
     2281 ccggggactg ggggtccctc tactccccag tctcccaggc tggcagcctg agtcccgccg
     2341 cgtcgctgga ggagcgaccc gggctgctgg gggccacctt ttccgcctgc ttgagcccag
     2401 gcagtctggc tttctcagat tttctgggct ccgctggctc cgctgctggt tctggcgaat
     2461 ttgactacaa agaccatgac ggtgattata aagatcatga catcgattac aaggatgacg
     2521 atgacaagga tatcaacacc ccgggaatta acctgatcaa ggaggacatg cgcgtgaagg
     2581 tgcacatgga gggcaacgtg aacggccacg ccttcgtgat cgagggcgag ggcaagggca
     2641 agccctacga gggcacccag accgccaacc tgaccgtgaa ggagggcgcc cccctgccct
     2701 tcagctacga catcctgacc accgccgtgc actacggcaa ccgggtgttc accaagtacc
     2761 ccgaggacat ccccgactac ttcaagcaga gcttccccga gggctacagc tgggagcgca
     2821 ccatgacctt cgaggacaag ggcatctgca ccatccgcag cgacatcagc ctggagggcg
     2881 actgcttctt ccagaacgtg cgcttcaagg gcaccaactt cccccccaac ggccccgtga
     2941 tgcagaagaa gaccctgaag tgggagccca gcaccgagaa gctgcacgtg cgcgacggcc
     3001 tgctggtggg caacatcaac atggccctgc tgctggaggg cggcggccac tacctgtgcg
     3061 acttcaagac cacctacaag gccaagaagg tggtgcagct gcccgacgcc cacttcgtgg
     3121 accaccgcat cgagatcctg ggcaacgaca gcgactacaa caaggtgaag ctgtacgagc
     3181 acgccgtggc ccgctacagc cccctgccca gccaggtgtg ggatatctac ccatacgatg
     3241 ttccagatta cgcttgaacc cagctttctt gtacaaagtg ggcccctctc cctccccccc
     3301 ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt
     3361 attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt
     3421 cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa
     3481 tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac
     3541 cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg
     3601 tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt
     3661 tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca
     3721 gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt
     3781 ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg
     3841 aaaaacacga tgataatatg gccacaacca tgaccgagta caagcccacg gtgcgcctcg
     3901 ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc
     3961 ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag
     4021 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg
     4081 ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga
     4141 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag
     4201 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct
     4261 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg
     4321 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct
     4381 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct
     4441 ggtgcatgac ccgcaagccc ggtgcctgac aactttatta tacatagttg atggccggcc
     4501 gcttcgagca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     4561 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     4621 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     4681 ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta gcggccgcgg
     4741 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4801 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4861 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4921 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4981 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5041 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctctcttcg
     5101 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5161 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5221 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5281 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5341 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     5401 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5461 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     5521 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     5581 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     5641 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     5701 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     5761 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     5821 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     5881 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     5941 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6001 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6061 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6121 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6181 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6241 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6301 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6361 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6421 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6481 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     6541 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     6601 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     6661 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     6721 aggcgtatca cgaggccctt tcgtcggcgc gccgcggccg c
//
