LOCUS       Exported                6854 bp ds-DNA     circular SYN 30-JAN-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6854)
  AUTHORS   Wythe Lab
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Jul 7, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6854
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        475..1653
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          706..1644
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     promoter        1670..1688
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1720..1768
                     /label=oJDW 2128
     protein_bind    1720..1744
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1776..1785
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             join(1782..2274,2533..2761)
                     /codon_start=1
                     /label=EGFP
                     /label=EGFP 5' half
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VKLQDPPQHRGRQRAARRPLPAEHPHRRRPRAAARQPLPEHPVRPEQRPQREARSHGPA
                     GVRDRRRDHSRHGRAVQV"
     intron          2275..2532
                     /label=Artificial intron and MCS
     intron          2275..2350
                     /label=Intron Total
     misc_feature    2347..2489
                     /label=Homology to Human HPRT
     primer_bind     complement(2748..2814)
                     /label=oJDW 2129
     protein_bind    complement(2790..2814)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2840..2881
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2891..2908
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    2931..2948
                     /label=BGH rev primers
     polyA_signal    2937..3161
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3207..3635
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        3649..3979
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     promoter        4027..4074
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4093..4491
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4649..4770
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    complement(4686..4705)
                     /label=SV40pA REVSeq
     primer_bind     complement(4815..4835)
                     /label=M13-rev
     protein_bind    4843..4859
                     /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(4867..4897)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4912..4933
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5203..5885)
                     /direction=LEFT
                     /label=ColE1 origin
     CDS             complement(5980..6840)
                     /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(join(6841..6854,1..91))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
       61 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
      121 tcgacggatc gggagatctc ccgatcccct atggtcgact ctcagtacaa tctgctctga
      181 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
      241 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
      301 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      361 ttgattattg actaggcttt tgcaaaaagc tttgcaaaga tggataaagt tttaaacaga
      421 gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa ggagtgcctc gtgaggctcc
      481 ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg
      541 gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc
      601 gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc
      661 gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtaag tgccgtgtgt
      721 ggttcccgcg ggcctggcct ctttacgggt tatggccctt gcgtgccttg aattacttcc
      781 acctggctgc agtacgtgat tcttgatccc gagcttcggg ttggaagtgg gtgggagagt
      841 tcgaggcctt gcgcttaagg agccccttcg cctcgtgctt gagttgaggc ctggcctggg
      901 cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc tgctttcgat
      961 aagtctctag ccatttaaaa tttttgatga cctgctgcga cgcttttttt ctggcaagat
     1021 agtcttgtaa atgcgggcca agatctgcac actggtattt cggtttttgg ggccgcgggc
     1081 ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg
     1141 ccaccgagaa tcggacgggg gtagtctcaa gctggccggc ctgctctggt gcctggcctc
     1201 gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc accagttgcg
     1261 tgagcggaaa gatggccgct tcccggccct gctgcaggga gctcaaaatg gaggacgcgg
     1321 cgctcgggag agcgggcggg tgagtcaccc acacaaagga aaagggcctt tccgtcctca
     1381 gccgtcgctt catgtgactc cacggagtac cgggcgccgt ccaggcacct cgattagttc
     1441 tcgagctttt ggagtacgtc gtctttaggt tggggggagg ggttttatgc gatggagttt
     1501 ccccacactg agtgggtgga gactgaagtt aggccagctt ggcacttgat gtaattctcc
     1561 ttggaatttg ccctttttga gtttggatct tggttcattc tcaagcctca gacagtggtt
     1621 caaagttttt ttcttccatt tcaggtgtcg tgaggaatta gcttggtact aatacgactc
     1681 actataggga gacccaagct ggctaggtaa gcttgatcaa caagtttgta caaaaaagca
     1741 ggcagctagc gctaccggac tcagatccac cggtcgccac catggtgagc aagggcgagg
     1801 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     1861 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     1921 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     1981 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     2041 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     2101 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     2161 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     2221 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgagtggat
     2281 cgggtaccga attccatgat cttttttttt tttttttttt tcccatgatc atcccgggga
     2341 tcctgcagat gcagtcagat ttctgaactg tctcagcaaa tggtaagtaa cttagagcta
     2401 gtaattaata acctgtcctt tgatttctga ttcagccaag aatggccata tttgggaaag
     2461 gcagatctgg agagtaacca cgttttcatg cagtctagac tgttaacgca gtgtttctct
     2521 aactctcagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     2581 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     2641 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     2701 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
     2761 agcggccgcg actctagacg cgtactagtg cccagctttc ttgtacaaag tggttgatct
     2821 agagggcccg cggttcgaag gtaagcctat ccctaaccct ctcctcggtc tcgattctac
     2881 gcgtaccggt catcatcacc atcaccattg agtttaaacc cgctgatcag cctcgactgt
     2941 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     3001 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     3061 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     3121 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     3181 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     3241 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3301 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3361 gggcatccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3421 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3481 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3541 tatctcggtc tattcttttg atttataagg gattttgggg atttcggcct attggttaaa
     3601 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     3661 gggtgtggaa agtccccagg ctccccaggc aggcagaagt atgcaaagca tgcatctcaa
     3721 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3781 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     3841 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     3901 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
     3961 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
     4021 gcacgtgttg acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg
     4081 aggaactaaa ccatggccaa gcctttgtct caagaagaat ccaccctcat tgaaagagca
     4141 acggctacaa tcaacagcat ccccatctct gaagactaca gcgtcgccag cgcagctctc
     4201 tctagcgacg gccgcatctt cactggtgtc aatgtatatc attttactgg gggaccttgt
     4261 gcagaactcg tggtgctggg cactgctgct gctgcggcag ctggcaacct gacttgtatc
     4321 gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct gcggacggtg tcgacaggtg
     4381 cttctcgatc tgcatcctgg gatcaaagcg atagtgaagg acagtgatgg acagccgacg
     4441 gcagttggga ttcgtgaatt gctgccctct ggttatgtgt gggagggcta agcacttcgt
     4501 ggccgaggag caggactgac acgtgctacg agatttcgat tccaccgccg ccttctatga
     4561 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     4621 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     4681 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     4741 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     4801 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     4861 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     4921 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4981 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     5041 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     5101 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     5161 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     5221 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     5281 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     5341 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     5401 cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     5461 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     5521 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     5581 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     5641 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     5701 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     5761 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     5821 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     5881 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     5941 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     6001 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     6061 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     6121 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     6181 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     6241 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     6301 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     6361 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     6421 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     6481 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     6541 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     6601 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     6661 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     6721 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     6781 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     6841 actcttcctt tttc
//
