LOCUS       Exported                6138 bp ds-DNA     circular SYN 30-JUN-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6138)
  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..6138
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     170..189
                     /label=oJDW 855 (CMV FWD)
     primer_bind     360..391
                     /label=FWD
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     exon            662..751
                     /label=chicken B-actin exon
     intron          752..1670
                     /label=chicken b-actin intron + rabbit b-globin intron
     misc_feature    1594..1616
                     /label=pCAGGS FWDSeq
     intron          1671..1723
                     /label=rabbit B-globin intron
     primer_bind     complement(1697..1723)
                     /label=Primer 1
     protein_bind    1737..1761
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    1774..1779
                     /label=Kozak
     CDS             1780..2156
                     /codon_start=1
                     /label=H. sapiens (human) Histone cluster 1, H2bb, H2B
                     /translation="MPEPSKSAPAPKKGSKKAITKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSS"
     misc_feature    1971
                     /label=should be a T(silent mutation)?
     CDS             2158..2172
                     /codon_start=1
                     /product="Gly Gly Ala Gly Gly Flexible Linker"
                     /label=G2AG2 Linker
                     /label=3X Flag GlyGlyAlaGlyGly
                     /note="Sabourin M et al., A flexible protein linker 
                     improves the function of epitope-tagged proteins in 
                     Saccharomyces cerevisiae (2007). Yeast."
                     /translation="GGAGG"
     CDS             2173..2214
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2215..2226
                     /codon_start=1
                     /label=MCS
                     /translation="GSTG"
     CDS             2227..2285
                     /codon_start=1
                     /label=GGGGS(x3)
                     /translation="EFDGGGGSGGGGSGGGGSP"
     CDS             2302..2988
                     /codon_start=1
                     /label=mScarlet3-H
                     /translation="MDSTEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LRVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYWKQSFPEGFKWERVMNFEDGGA
                     VSVAQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDVVLKGDIKH
                     ALRLKDGGRYLADFKTTYRAKKPVQMPGAFNIDRKLDITSHNEDYTVVEQYERSVARHS
                     TGGSGGS"
     regulatory      2302..2305
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(3045..3069)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    3226..3281
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3640..3656)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3664..3680
                     /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(3688..3718)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3733..3754
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3812..4008
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3859..3994
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4014..4148
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    complement(4051..4070)
                     /label=SV40pA REVSeq
     rep_origin      complement(4387..4975)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5146..6006)
                     /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(6007..6111)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
     1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     1681 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcctcgag gatatcacaa
     1741 gtttgtacaa aaaagcaggc tactagtgct agcgccacca tgcctgaacc ctctaagtct
     1801 gctccagccc ctaaaaaggg ttctaagaag gctatcacta aggcgcagaa gaaggatggt
     1861 aagaagcgta agcgcagccg caaggagagc tattctatct atgtgtacaa ggttctgaag
     1921 caggtccacc ccgacaccgg catctcatcc aaggccatgg ggatcatgaa ctccttcgtc
     1981 aacgacatct tcgagcgcat cgcgggcgag gcttctcgcc tggctcacta caataagcgc
     2041 tcgaccatca cctccaggga gattcagacg gctgtgcgcc tgctgctgcc tggggagctg
     2101 gctaagcatg ctgtgtccga gggcactaag gcagttacca agtacactag ctctaagggt
     2161 ggtgctggtg gtggtaagcc tatccctaac cctctcctcg gtctcgattc tacgggatcc
     2221 accggtgaat tcgatggtgg aggcggttca ggcggaggtg gctctggcgg tggcggatcg
     2281 ccatcaggta ccatggtgag catggatagc accgaggcag tgatcaagga gttcatgcgg
     2341 ttcaaggtgc acatggaggg ctccatgaac ggccacgagt tcgagatcga gggcgagggc
     2401 gagggccgcc cctacgaggg cacccagacc gccaagctga gggtgaccaa gggtggcccc
     2461 ctgcccttct cctgggacat cctgtcccct cagttcatgt acggctccag ggccttcacg
     2521 aagcaccccg ccgacatccc cgactactgg aagcagtcct tccccgaggg cttcaagtgg
     2581 gagcgcgtga tgaacttcga ggacggcggc gccgtgtccg tggcccagga cacctccctg
     2641 gaggacggca ccctgatcta caaggtgaag ctccgcggca ccaacttccc tcctgacggc
     2701 cccgtaatgc agaagaagac aatgggctgg gaagcatcca ccgagcggtt gtaccccgag
     2761 gacgtcgtgc tgaagggcga cattaagcat gccctgcgcc tgaaggacgg cggccggtac
     2821 ctggcggact tcaagaccac ctacagggcc aagaagcccg tgcagatgcc cggcgccttc
     2881 aacatcgacc gcaagttgga catcacatcc cacaacgagg actacaccgt ggtggaacag
     2941 tacgaacgct ccgtggcccg ccactccacc ggcggctccg gtggctccta actataatga
     3001 ggcgcgccgt cgacgttaac acgcgttaat gatacgtatc tagaacccag ctttcttgta
     3061 caaagtggtg atatcgcggc cgcactcctc aggtgcaggc tgcctatcag aaggtggtgg
     3121 ctggtgtggc caatgccctg gctcacaaat accactgaga tctttttccc tctgccaaaa
     3181 attatgggga catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta
     3241 ttttcattgc aatagtgtgt tggaattttt tgtgtctctc actcggaagg acatatggga
     3301 gggcaaatca tttaaaacat cagaatgagt atttggttta gagtttggca acatatgcca
     3361 tatgctggct gccatgaaca aaggtggcta taaagaggtc atcagtatat gaaacagccc
     3421 cctgctgtcc attccttatt ccatagaaaa gccttgactt gaggttagat tttttttata
     3481 ttttgttttg tgttattttt ttctttaaca tccctaaaat tttccttaca tgttttacta
     3541 gccagatttt tcctcctctc ctgactactc ccagtcatag ctgtccctct tctcttatga
     3601 agatccctcg acctgcagcc caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     3661 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     3721 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     3781 ccagtcggga aacctgtcgt gccagcggat ccgcatctca attagtcagc aaccatagtc
     3841 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     3901 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     3961 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctaacttgt
     4021 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     4081 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4141 tctggatccg ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     4201 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     4261 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     4321 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4381 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4441 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4501 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4561 gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4621 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4681 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4741 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     4801 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     4861 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     4921 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     4981 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5041 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     5101 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     5161 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     5221 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     5281 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     5341 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5401 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5461 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5521 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5581 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     5641 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     5701 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     5761 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     5821 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     5881 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     5941 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6001 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6061 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     6121 ccgaaaagtg ccacctga
//
