LOCUS       Exported                7080 bp ds-DNA     circular SYN 17-JAN-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7080)
  AUTHORS   MIT
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Jan 21, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7080
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     109..128
                     /label=oSEQ054 pMXs-fwd-seq
     primer_bind     complement(198..211)
                     /label=oSEQ013 flipped_WPRE_rev
     protein_bind    222..246
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(223..241)
                     /label=?oSEQ049 attR1-rev
     CDS             285..296
                     /codon_start=1
                     /label=uORF
                     /note="/created_by=User"
                     /translation="TMG"
     CDS             297..308
                     /codon_start=1
                     /label=uORF
                     /note="/created_by=User"
                     /translation="TMG"
     CDS             309..320
                     /codon_start=1
                     /label=uORF
                     /note="/created_by=User"
                     /translation="TMG"
     primer_bind     330..360
                     /label=gg_N-HA_fwd
     primer_bind     331..352
                     /label=Ngn2_1_fwd
     misc_feature    335..343
                     /label=Kozak sequence
                     /note="Partial (missing last G) vertebrate consensus 
                     sequence for strong initiation of translation (Kozak, 
                     1987)"
     CDS             341..1129
                     /codon_start=1
                     /product="Mouse Ngn2 "
                     /label=mNgn2
                     /translation="MFVKSETLELKEEEEVLMLLGSASPASATLTPMSSSADEEEDEEL
                     RRPGSARGQRGAEAGQGVQGSPASGAGGCRPGRLLGLMHECKRRPSRSRAVSRGAKTAE
                     TVQRIKKTRRLKANNRERNRMHNLNAALDALREVLPTFPEDAKLTKIETLRFAHNYIWA
                     LTETLRLADHCAGAGGLQGALFTEAVLLSPGAALGASGDSPSPPSSWSCTNSPASSSNS
                     TSPYSCTLSPASPGSDVDYWQPPPPEKHRYAPHLPLARDCI"
     primer_bind     341..362
                     /label=Ngn2_2_fwd
     primer_bind     341..362
                     /label=Ngn2_3_fwd
     primer_bind     complement(1108..1125)
                     /label=gg_N-HA_rev
     primer_bind     complement(1112..1131)
                     /label=Ngn2_1_rev
     primer_bind     complement(1112..1131)
                     /label=Ngn2_2_rev
     primer_bind     complement(1114..1129)
                     /label=Ngn2_3_rev
     primer_bind     1122..1149
                     /label=gg_HA_fwd
     CDS             1169..1267
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=3xHA
     CDS             1169..1195
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1205..1231
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1241..1267
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(1249..1271)
                     /label=Ngn2H_1_rev
     primer_bind     complement(1249..1269)
                     /label=Ngn2H_2_rev
     primer_bind     complement(1249..1267)
                     /label=Ngn2H_3_rev
     primer_bind     1268..1290
                     /label=oKL166
     primer_bind     1268..1289
                     /label=oKL141
     primer_bind     1268..1288
                     /label=P2A_fwd
     CDS             1277..1333
                     /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"
     primer_bind     complement(1316..1333)
                     /label=P2A_rev
     primer_bind     complement(1318..1339)
                     /label=ggNgn2-P2A_rev
     primer_bind     1324..1352
                     /label=P2A-mRuby2_fwd
     CDS             1334..2044
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     primer_bind     1334..1352
                     /label=ggmRuby2_fwd
     primer_bind     complement(2027..2051)
                     /label=ggmRuby2_rev
     protein_bind    complement(2061..2085)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2102..2690
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2268..2287)
                     /label=oSEQ034 WPRE_rev
     primer_bind     2525..2545
                     /label=oSEQ048 phage-dest-fwd-2
     CDS             complement(2573..2584)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     2687..2704
                     /label=oSEQ013 flipped_WPRE_rev
     primer_bind     complement(2729..2747)
                     /label=oSEQ055 pMXs-rev-seq
     LTR             2870..3339
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     rep_origin      complement(3640..4228)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3720..3739)
                     /label=oSEQ067 pBR322-ori_fwd
     CDS             complement(4392..5252)
                     /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"
     primer_bind     5015..5034
                     /label=oSEQ066 Amp_rev
     primer_bind     complement(5228..5252)
                     /label=oSEQ083 Amp-fwd
     promoter        complement(5253..5357)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5299..5320
                     /label=oSEQ095 oKL157
     LTR             5387..5980
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    6043..6400
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             6459..6875
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     complement(6653..6672)
                     /label=oSEQ082 pMX-gag-rev
     primer_bind     6653..6672
                     /label=oSEQ096 pMX-gag_fwd
     misc_feature    join(6885..7080,1..179)
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
ORIGIN
        1 tcggcgacac agtgtgggtc cgccgacacc agactaagaa cctagaacct cgctggaaag
       61 gaccttacac agtcctgctg accaccccca ccgccctcaa agtagacggc atcgcagctt
      121 ggatacacgc cgcccacgtg aaggctgccg accccggggg tggaccatcc tctagactgc
      181 cggatctagc tagttaatcg acggtatcga taagctatca aacaagtttg tacaaaaaag
      241 caggctccac catgggaacc aattcagtcg agtcgactgg atccaccatg ggttgaacca
      301 tgggttgaac catgggttga gatccggtac tactgccacc atgttcgtca aatctgagac
      361 tctggagttg aaggaggaag aggaggtact gatgctgctg ggctcggctt ccccggcctc
      421 ggcgaccctg accccgatgt cctccagcgc ggacgaggag gaggacgagg agctgcgccg
      481 gccgggctcc gcgcgtgggc agcgtggagc ggaagccggg cagggggtgc agggcagtcc
      541 ggcgtcgggt gccgggggtt gccggccagg gcggctgctg ggcctgatgc acgagtgcaa
      601 gcgtcgcccg tcgcgctcac gggccgtctc ccgaggtgcc aagacggcgg agacggtgca
      661 gcgcatcaag aagacccgca ggctcaaggc caacaaccgc gagcgcaacc gcatgcacaa
      721 cctaaacgcc gcgctggacg cgctgcgcga ggtgctgccc accttccccg aggatgccaa
      781 gctcacgaag atcgagacgc tgcgcttcgc ccacaattac atctgggcgc tcaccgagac
      841 tctgcgcctg gcggaccact gcgccggcgc cggtggcctc cagggggcgc tcttcacgga
      901 ggcggtgctc ctgagcccgg gagctgcgct cggcgccagc ggggacagcc cttctccacc
      961 ttcctcctgg agctgcacca acagcccggc gtcatcctcc aactccacgt ccccatacag
     1021 ctgcacttta tcgcccgcta gccccgggtc agacgtggac tactggcagc ccccacctcc
     1081 ggagaagcat cgttatgcgc ctcacctgcc cctcgccagg gactgtatcg ggtctggctc
     1141 cggatcaagt ggtggcagcg gatcaggata tccatacgat gtaccagact atgcgggatc
     1201 gggttacccc tacgacgtcc ccgactacgc cggctccggc tacccctacg acgtccctga
     1261 ttatgctgga agcggagcta ctaacttcag cctgctgaag caggctggag acgtggagga
     1321 gaaccctgga cctatggtgt ctaagggcga agagctgatc aaggaaaata tgcgtatgaa
     1381 ggtggtcatg gaaggttcgg tcaacggcca ccaattcaaa tgcacaggtg aaggagaagg
     1441 caatccgtac atgggaactc aaaccatgag gatcaaagtc atcgagggag gacccctgcc
     1501 atttgccttt gacattcttg ccacgtcgtt catgtatggc agccgtactt ttatcaagta
     1561 cccgaaaggc attcctgatt tctttaaaca gtcctttcct gagggtttta cttgggaaag
     1621 agttacgaga tacgaagatg gtggagtcgt caccgtcatg caggacacca gccttgagga
     1681 tggctgtctc gtttaccacg tccaagtcag aggggtaaac tttccctcca atggtcccgt
     1741 gatgcagaag aagaccaagg gttgggagcc taatacagag atgatgtatc cagcagatgg
     1801 tggtctgagg ggatacactc atatggcact gaaagttgat ggtggtggcc atctgtcttg
     1861 ctctttcgta acaacttaca ggtcaaaaaa gaccgtcggg aacatcaaga tgcccggtat
     1921 ccatgccgtt gatcaccgcc tggaaaggtt agaggaaagt gacaatgaaa tgttcgtagt
     1981 acaacgcgaa cacgcagttg ccaagttcgc cgggcttggt ggtgggatgg acgagctgta
     2041 caagtaacgc tgatatctag acccagcttt cttgtacaaa gtggttcgat agcttatcga
     2101 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     2161 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     2221 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     2281 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     2341 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     2401 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     2461 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     2521 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     2581 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     2641 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg
     2701 tcgacgataa aataaaagat tttatttagt ctccagaaaa aggggggaat gaaagacccc
     2761 acctgtaggt ttggcaagct agcttaagta acgccatttt gcaaggcatg gaaaaataca
     2821 taactgagaa tagagaagtt cagatcaagg tcaggaacag atggaacagc tgaatatggg
     2881 ccaaacagga tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatgga
     2941 acagctgaat atgggccaaa caggatatct gtggtaagca gttcctgccc cggctcaggg
     3001 ccaagaacag atggtcccca gatgcggtcc agccctcagc agtttctaga gaaccatcag
     3061 atgtttccag ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga actaaccaat
     3121 cagttcgctt ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata aaagagccca
     3181 caacccctca ctcggggcgc cagtcctccg attgactgag tcgcccgggt acccgtgtat
     3241 ccaataaacc ctcttgcagt tgcatccgac ttgtggtctc gctgttcctt gggagggtct
     3301 cctctgagtg attgactacc cgtcagcggg ggtctttcac atgcagcatg tatcaaaatt
     3361 aatttggttt tttttcttaa gtatttacat taaatggcca tagttgcatt aatgaatcgg
     3421 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     3481 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     3541 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     3601 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3661 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3721 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3781 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3841 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3901 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3961 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     4021 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     4081 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     4141 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     4201 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     4261 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     4321 cttcacctag atccttttgc ggccggccgc aaatcaatct aaagtatata tgagtaaact
     4381 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4441 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     4501 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     4561 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     4621 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     4681 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     4741 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     4801 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     4861 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     4921 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     4981 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     5041 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     5101 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     5161 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     5221 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     5281 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     5341 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgcataatg aaagacccca
     5401 cctgtaggtt tggcaagcta gcttaagtaa cgccattttg caaggcatgg aaaaatacat
     5461 aactgagaat agaaaagttc agatcaaggt caggaacaga tggaacagct gaatatgggc
     5521 caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga acagatggaa
     5581 cagctgaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcagggc
     5641 caagaacaga tggtccccag atgcggtcca gccctcagca gtttctagag aaccatcaga
     5701 tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc
     5761 agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa aagagcccac
     5821 aacccctcac tcggcgcgcc agtcctccga ttgactgagt cgcccgggta cccgtgtatc
     5881 caataaaccc tcttgcagtt gcatccgact tgtggtctcg ctgttccttg ggagggtctc
     5941 ctctgagtga ttgactaccc gtcagcgggg gtctttcatt tgggggctcg tccgggatcg
     6001 ggagacccct gcccagggac caccgaccca ccaccgggag gtaagctggc cagcaactta
     6061 tctgtgtctg tccgattgtc tagtgtctat gactgatttt atgcgcctgc gtcggtacta
     6121 gttagctaac tagctctgta tctggcggac ccgtggtgga actgacgagt tcggaacacc
     6181 cggccgcaac cctgggagac gtcccaggga cttcgggggc cgtttttgtg gcccgacctg
     6241 agtccaaaaa tcccgatcgt tttggactct ttggtgcacc cccctaatag gagggatatg
     6301 tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt
     6361 cggtttggga ccgaagccgc gccgcgcgtc ttgtctgctg cagcatcgtt ctgtgttgtc
     6421 tctgtctgac tgtgtttctg tatttgtctg aaaattaggg ccagactgtt accactccct
     6481 taagtttgac cttaggtcac tggaaagatg tcgagcggat cgctcacaac cagtcggtag
     6541 atgtcaagaa gagacgttgg gttaccttct gctctgcaga atggccaacc tttaacgtcg
     6601 gatggccgcg agacggcacc tttaaccgag acctcatcac ccaggttaag atcaaggtct
     6661 tttcacctgg cccgcatgga cacccagacc aggtccccta catcgtgacc tgggaagcct
     6721 tggcttttga cccccctccc tgggtcaagc cctttgtaca ccctaagcct ccgcctcctc
     6781 ttcctccatc cgccccgtct ctcccccttg aacctcctcg ttcgaccccg cctcgatcct
     6841 ccctttatcc agccctcact ccttctctag gcgcccccat atggccatat gagatcttat
     6901 atggggcacc cccgcccctt gtaaacttcc ctgaccctga catgacaaga gttactaaca
     6961 gcccctctct ccaagctcac ttacaggctc tctacttagt ccagcacgaa gtctggagac
     7021 ctctggcggc agcctaccaa gaacaactgg accgaccggt ggtacctcac ccttaccgag
//
