LOCUS       Exported                5628 bp ds-DNA     circular SYN 13-OCT-2023
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5628)
  AUTHORS   Jiten Doshi
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Nov 4, 2024 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5628
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    45..88
                     /label=T7
                     /label=nonstandard type: misc_feature_
     terminator      complement(46..92)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    110..158
                     /note="synthetic polyadenylation signal"
     primer_bind     189..205
                     /label=PR-JD21
                     /note="fp to amplify ef1a promoter from pJD02 to transfer 
                     to pJD10; Gibson assembly"
     misc_feature    190..1373
                     /label=EF1a
                     /label=nonstandard type: misc_feature_
     intron          426..1364
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1407..1426
                     /label=PR-JD47
                     /note="sequencing primer pJD01"
     primer_bind     complement(1449..1469)
                     /label=PR-JD22
                     /note="rp to amplify ef1a promoter from pJD02 to transfer 
                     to pJD10; Gibson assembly"
     misc_feature    1470..1583
                     /label=insert
                     /label=nonstandard type: misc_feature_
     misc_feature    1470..1569
                     /label=synthetic 5' UTR
                     /note="/from pJD48; pRA16"
     primer_bind     1470..1493
                     /label=PR-JD58
                     /note="syn5utr reverse primer for part2 golden gate 
                     verification; (150bp product)"
     regulatory      1579..1583
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     misc_feature    1579..1583
                     /gene="Kozak sequence"
                     /label=Kozak sequence
     primer_bind     1584..1603
                     /label=JD407
     CDS             1585..2268
                     /codon_start=1
                     /product="33K"
                     /label=33K
                     /protein_id="AP_000215.1"
                     /translation="MAPKKKLQLPPPPTDEEEYWDSQAEEVLDEEEEDMMEDWESLDEE
                     ASEVEEVSDETPSPSVAFPSPAPQKSATGSSMATTSAPQAPPALPVRRPNRRWDTTGTR
                     AAHTAPAAAAATAAATQKQRRPDSKTLTKPKKSTAAAAAGGGALRLAPNEPVSTRELRN
                     RIFPTLYAIFQQSRGQEQELKIKNRSLRSLTRSCLYHKSEDQLRRTLEDAEALFSKYCA
                     LTLKD"
     misc_feature    1585..2165
                     /label=intron
     CDS             1585..1900
                     /codon_start=1
                     /product="22K"
                     /label=22K
                     /protein_id="AP_000216.1"
                     /translation="MAPKKKLQLPPPPTDEEEYWDSQAEEVLDEEEEDMMEDWESLDEE
                     ASEVEEVSDETPSPSVAFPSPAPQKSATGSSMATTSAPQAPPALPVRRPNRRWDTTGTR
                     A"
     CDS             1585..1874
                     /codon_start=1
                     /product="100K"
                     /label=100K
                     /protein_id="AP_000214.1"
                     /translation="GTQKEAAAAAATHGRGGILGQSGRGGFGRGGGGHDGRLGEPRRGS
                     FRGRRGVRRNTVTLGRIPLAGAPEIGNRFQHGYNLRSSGAAGTARSPTQP"
     primer_bind     1587..1607
                     /label=JD262
     primer_bind     1718..1736
                     /label=JD196
                     /note="e2a gene sequencing primer"
     primer_bind     complement(1719..1731)
                     /label=PR-JD22
                     /note="rp to amplify ef1a promoter from pJD02 to transfer 
                     to pJD10; Gibson assembly"
     primer_bind     complement(1803..1822)
                     /label=JD195
                     /note="e2a gene sequencing primer"
     primer_bind     complement(1857..1874)
                     /label=JD263
     misc_feature    1875..1890
                     /label=fp qPCR
     primer_bind     complement(1883..1904)
                     /label=JD408
     misc_feature    1900
                     /label=rp qPCR
     CDS             1901..1967
                     /codon_start=1
                     /product="22K"
                     /label=22K
                     /protein_id="AP_000216.1"
                     /translation="PYCTGGSGSNSSGHTEAKATG"
     primer_bind     1901..1918
                     /label=JD409
     primer_bind     complement(1946..1967)
                     /label=JD244
     misc_feature    2160..2168
                     /label=8.48
     primer_bind     complement(2169..2186)
                     /label=JD132
                     /note="use for CMV-E2A cloning; use instead of JD115 for 
                     pJD78(2) construct as template"
     primer_bind     complement(2169..2186)
                     /label=JD145
                     /note="fp; template pJD90; for part2 of CMV-E2A(mod5utr) 
                     cloning; part3 similar to pJD78; use JD116 as rp"
     misc_feature    2187..2264
                     /label=E2A_promoter??
     primer_bind     complement(2247..2268)
                     /label=JD245
     misc_feature    join(2265..5628,1..1469)
                     /label=vector_backbone-835-5669
                     /label=nonstandard type: misc_feature_
     misc_feature    2270..2296
                     /label=junk bacterial sequence??
                     /note="blast shows match to bacterial sequences!"
     misc_feature    2297..2530
                     /label=SV40_PA
                     /label=nonstandard type: misc_feature_
     primer_bind     complement(2297..2316)
                     /label=PR-JD48
                     /note="sequencing primer pJD01"
     misc_feature    2369..2374
                     /label=pA signal (bottom strand)
     primer_bind     2378..2397
                     /label=SV40pA-R
                     /note="Genewiz primer collection"
     misc_feature    2398..2403
                     /label=pA signal (bottom strand)
     misc_feature    2423..2428
                     /label=pA signal
     primer_bind     2468..2484
                     /label=EGFP-C-REV
                     /note="Genewiz primer collection"
     terminator      2549..2595
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      2625..2761
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2806..2877
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(3103..3691)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    3103..3691
                     /label=colE1_high_copy
                     /label=nonstandard type: misc_feature_
     primer_bind     complement(3148..3167)
                     /label=PR-JD29
                     /note="pJD11 rtTA CDS, pA sequencing primer"
     primer_bind     3709..3727
                     /label=PR-JD32
                     /note="pJD02/03 sequencing primer"
     CDS             4437..5297
                     /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"
     misc_feature    4437..5297
                     /label=ampR
                     /label=nonstandard type: misc_feature_
     terminator      5310..5446
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    join(5598..5628,1..41)
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
ORIGIN
        1 cttccctcta gtggtcataa cagcagcttc agctacctct caattcaaaa aacccctcaa
       61 gacccgttta gaggccccaa ggggttatgc tatcaatcgt tgcgttacac acacaaaaaa
      121 ccaacacaca tccatcttcg atggatagcg attttattat ctaactgctg atcgagtgta
      181 gccagatctc gtgaggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc
      241 ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg
      301 taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac
      361 cgtatataag tgcactagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa
      421 cacaggtaag tgccgtgtgt ggttcccgcg ggcctggcct ctttacgggt tatggccctt
      481 gcgtgccttg aattacttcc acctggctgc agtacgtgat tcttgatccc gagcttcggg
      541 ttggaagtgg gtgggagagt tcgtggcctt gcgcttaagg agccccttcg cctcgtgctt
      601 gagttgtggc ctggcctggg cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg
      661 cctgtctcgc tgctttcgat aagtctctag ccatttaaaa tttttgatga cctgctgcga
      721 cgcttttttt ctggcaagat agtcttgtaa atgcgggcca agatcagcac actggtattt
      781 cggtttttgg ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga
      841 ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa gctgcccggc
      901 ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg
      961 cccggtcggc accagttgcg tgagcggaaa gatggccgct tcccggccct gctgcaggga
     1021 gcacaaaatg gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc acacaaagga
     1081 aaagggcctt tccgtcctca gccgtcgctt catgtgactc cacggagtac cgggcgccgt
     1141 ccaggcacct cgattagttc tccagctttt ggagtacgtc gtctttaggt tggggggagg
     1201 ggttttatgc gatggagttt ccccacactg agtgggtgga gactgaagtt aggccagctt
     1261 ggcacttgat gtaattctcc ttggaatttg ccctttttga gtttggatct tggttcattc
     1321 tcaagcctca gacagtggtt caaagttttt ttcttccatt tcaggtgtcg tgaaaactac
     1381 ccctaaaagc caaaagatct ttgtcgatcc taccatccac tcgacacacc cgccagcggc
     1441 cgctgccaag cttccgagct ctcgaattca tcttggctga ggaatcttct aacaatttag
     1501 agcttaaaaa cgcccacgag gcggagaacg aaatatccag agagacgtta gaaacgttca
     1561 aaaacgttca ccggtagggc caccatggca cccaaaaaga agctgcagct gccgccgcca
     1621 cccacggacg aggaggaata ctgggacagt caggcagagg aggttttgga cgaggaggag
     1681 gaggacatga tggaagactg ggagagccta gacgaggaag cttccgaggt cgaagaggtg
     1741 tcagacgaaa caccgtcacc ctcggtcgca ttcccctcgc cggcgcccca gaaatcggca
     1801 accggttcca gcatggctac aacctccgct cctcaggcgc cgccggcact gcccgttcgc
     1861 cgacccaacc gtagatggga caccactgga accagggccg cccatactgc accggcggca
     1921 gcggcagcaa cagcagcggc cacacagaag caaaggcgac cggatagcaa gactctgaca
     1981 aagcccaaga aatccacagc ggcggcagca gcaggaggag gagcgctgcg tctggcgccc
     2041 aacgaacccg tatcgacccg cgagcttaga aacaggattt ttcccactct gtatgctata
     2101 tttcaacaga gcaggggcca agaacaagag ctgaaaataa aaaacaggtc tctgcgatcc
     2161 ctcacccgca gctgcctgta tcacaaaagc gaagatcagc ttcggcgcac gctggaagac
     2221 gcggaggctc tcttcagtaa atactgcgcg ctgactctta aggactagct tgactgactg
     2281 agatacagcg taccttcagc tcacagacat gataagatac attgatgagt ttggacaaac
     2341 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     2401 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     2461 gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg
     2521 tggtattggc ccatctctat cggtatcgta gcataacccc ttggggcctc taaacgggtc
     2581 ttgaggggtt ttttgtgccc ctcgggccgg attgctatct accggcattg gcgcagaaaa
     2641 aaatgcctga tgcgacgctg cgcgtcttat actcccacat atgccagatt cagcaacgga
     2701 tacggcttcc ccaacttgcc cacttccata cgtgtcctcc ttaccagaaa tttatcctta
     2761 aggtcgtcag ctatcctgca ggcgatctct cgatttcgat caagacattc ctttaatggt
     2821 cttttctgga caccactagg ggtcagaagt agttcatcaa actttcttcc ctccctaatc
     2881 tcattggtta ccttgggcta tcgaaactta attaaccagt caagtcagct acttggcgag
     2941 atcgacttgt ctgggtttcg actacgctca gaattgcgtc agtcaagttc gatctggtcc
     3001 ttgctattgc acccgttctc cgattacgag tttcatttaa atcatgtgag caaaaggcca
     3061 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     3121 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     3181 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     3241 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     3301 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     3361 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     3421 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     3481 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     3541 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     3601 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     3661 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     3721 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     3781 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     3841 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     3901 ctgtctattt cgttcatcca tagttgcatt taaatttccg aactctccaa ggccctcgtc
     3961 ggaaaatctt caaacctttc gtccgatcca tcttgcaggc tacctctcga acgaactatc
     4021 gcaagtctct tggccggcct tgcgccttgg ctattgcttg gcagcgccta tcgccaggta
     4081 ttactccaat cccgaatatc cgagatcggg atcacccgag agaagttcaa cctacatcct
     4141 caatcccgat ctatccgaga tccgaggaat atcgaaatcg gggcgcgcct ggtgtaccga
     4201 gaacgatcct ctcagtgcga gtctcgacga tccatatcgt tgcttggcag tcagccagtc
     4261 ggaatccagc ttgggaccca ggaagtccaa tcgtcagata ttgtactcaa gcctggtcac
     4321 ggcagcgtac cgatctgttt aaacctagat attgatagtc tgatcggtca acgtataatc
     4381 gagtcctagc ttttgcaaac atctatcaag agacaggatc agcaggaggc tttcgcatga
     4441 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     4501 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcgcgag
     4561 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     4621 aacgctttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     4681 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     4741 agtattcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     4801 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgattggag
     4861 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     4921 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     4981 tagcaatggc aacaaccttg cgtaaactat taactggcga actacttact ctagcttccc
     5041 ggcaacagtt gatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     5101 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     5161 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     5221 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     5281 tgattaagca ttggtaaccg attctaggtg cattggcgca gaaaaaaatg cctgatgcga
     5341 cgctgcgcgt cttatactcc cacatatgcc agattcagca acggatacgg cttccccaac
     5401 ttgcccactt ccatacgtgt cctccttacc agaaatttat ccttaagatc ccgaatcgtt
     5461 taaactcgac tctggctcta tcgaatctcc gtcgtttcga gcttacgcga acagccgtgg
     5521 cgctcatttg ctcgtcgggc atcgaatctc gtcagctatc gtcagcttac ctttttggca
     5581 gcgatcgcgg ctcccgacat cttggaccat tagctccaca ggtatctt
//
