LOCUS       sequence-386598-        6460 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ""Insert(low)-pEF1s(orig)"" reporter plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6460)
  AUTHORS   Ma Y, Budde MW, Zhu J, Elowitz MB
  TITLE     Tuning Methylation-Dependent Silencing Dynamics by Synthetic 
            Modulation of CpG Density.
  JOURNAL   ACS Synth Biol. 2023 Aug 12. doi: 10.1021/acssynbio.3c00078.
  PUBMED    37572041
REFERENCE   2  (bases 1 to 6460)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth 
            Biol. 2023 Aug 12. doi: 10.1021/acssynbio.3c00078."
COMMENT     Sequence Label: vYM015_phiC31-nTRE-Ins(low)-pEF1s(orig)-H2b-Ci...
FEATURES             Location/Qualifiers
     source          1..6460
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      77..665
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     566..585
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    849..983
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    1086..1207
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    1244..1313
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     CDS             1579..2382
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1642..1661)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2252..2271
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(2414..2431)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2420..2644
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    2717..2735
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2753..2771
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2789..2807
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2825..2843
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2861..2879
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        3255..3466
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             3479..3747
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             3833..4210
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      4229..4238
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4235..4237,4238..4240,4241..4951)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4280..4301)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4541..4560)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4888..4909
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5002..5021)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5003..5021)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    5295..5416
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        5442..5505
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5506..5574,5575..6366)
                     /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     complement(5724..5743)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
       61 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
      121 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      181 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
      241 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      301 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      361 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      421 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      481 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      541 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      601 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      661 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      721 acatgttctt tcctgcggta ccgatcataa tcagccatac cacatttgta gaggttttac
      781 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
      841 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
      901 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
      961 atgtatctta tcatgtctgg atcataatca gccataccac atttgtagag gttttacttg
     1021 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     1081 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     1141 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     1201 tatcttatca tgtctgctcg aggtcgacga tgtaggtcac ggtctcgaag ccgcggtgcg
     1261 ggtgccaggg cgtgcccttg ggctccccgg gcgcgtactc cacctcaccc atctggtcca
     1321 tcatgatgaa cgggtcgagg tggcggtagt tgatcccggc gaacgcgcgg cgcaccggga
     1381 agccctcgcc ctcgaaaccg ctgggcgcgg tggtcacggt gagcacggga cgtgcgacgg
     1441 cgtcggcggg tgcggatacg cggggcagcg tcagcgggtt ctcgacggtc acggcgggca
     1501 tgtcgacggt atcgataagc ttacgcgtgc tagcgcatgc agatctacta gtcggccgga
     1561 taaagatatc aactcgagat gggatcggcc attgaacaag atggattgca cgcaggttct
     1621 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     1681 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     1741 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     1801 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     1861 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     1921 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     1981 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     2041 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     2101 gccaggctca aggcgcgcat gcccgacggc gatgatctcg tcgtgaccca tggcgatgcc
     2161 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     2221 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     2281 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     2341 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gaggggatca attctctaga
     2401 gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     2461 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     2521 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     2581 gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggatgc ggtgggctct
     2641 atggcttctg aggcggaaag aaccagctgg gggctagcaa agcctaggaa cagctgcata
     2701 tagcactgca gtttactccc tatcagtgat agagagtcaa aaggccatgc cctccctatc
     2761 agtgatagag agagcagact caaggtggtc cctatcagtg atagagagat tgagcaagag
     2821 ggcctcccta tcagtgatag agattgatcc agtcagggcc tccctatcag tgatagagaa
     2881 gctatgtgca ggtagggctg tacagtggga atgcacgtaa gctgctatac tataggcctt
     2941 ggccttcagc agcaagcaaa agcaggagca ggcgccagct ggccaaggcg agagctgcct
     3001 ggcaggcgag gccctgctgc caattgggtg tgtgcgccta agcagcttgc tatgcaagct
     3061 ttatttgctt gcaggtgtgc cttgcagccc taagcagcag gccaagtgtg ccaaaggagc
     3121 cttaggcgcc atttgcagtg caggagagtg ctggcgcctg agcgtggtgc cagcatggag
     3181 ctaggcgccc agctggtggc ctagctaggc ctatggcctt gtagggatcg gcgatcgctc
     3241 cggtgcccgt cagcgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
     3301 ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
     3361 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
     3421 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacagctga agcttcgagg
     3481 ggctcgcatc tctccttcac gcgcccgccg ccctacctga ggccgccatc cacgccggtt
     3541 gagtcgcgtt ctgccgcctc ccgcctgtgg tgcctcctga actgcgtccg ccgtctaggt
     3601 aagtttaaag ctcaggtcga gaccgggcct ttgtccggcg ctcccttgga gcctacctag
     3661 actcagccgg ctctccacgc tttgcctgac cctgcttgct caactctacg tctttgtttc
     3721 gttttctgtt ctgcgccgtt acagatccaa gctgtgacct gggcctacac ctggccgctc
     3781 tagcccgggc ggatcccccg ggctgcagga attcgatatc aagcttgcca ccatgccaga
     3841 gccagcgaag tctgctcccg ccccgaaaaa gggctccaag aaggcggtga ctaaggcgca
     3901 gaagaaaggc ggcaagaagc gcaagcgcag ccgcaaggag agctattcca tctatgtgta
     3961 caaggttctg aagcaggtcc accctgacac cggcatttcg tccaaggcca tgggcatcat
     4021 gaattcgttt gtgaacgaca ttttcgagcg catcgcaggt gaggcttccc gcctggcgca
     4081 ttacaacaag cgctcgacca tcacctccag ggagatccag acggccgtgc gcctgctgct
     4141 gcctggggag ttggccaagc acgccgtgtc cgagggtact aaggccatca ccaagtacac
     4201 cagcgctaag gatccccggg taccggtcgc caccatggtg agcaagggcg aggagctgtt
     4261 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     4321 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     4381 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccttcg gctacggcct
     4441 gatgtgcttc gcccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     4501 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     4561 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     4621 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     4681 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     4741 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     4801 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agctaccagt ccgccctgag
     4861 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     4921 gatcactctc ggcatggacg agctgtacaa gtaggtcgac ctcgaggggg ggcccggtac
     4981 caggtaagtg tacccaattc gccctatagt gagtcgtatt acaattcact cgatcgccct
     5041 tcccaacagt tgcgcagcct gaatggcgaa tggagatcca atttttaagt gtataatgtg
     5101 ttaaactact gattctaatt gtttgtgtat tttagattca cagtcccaag gctcatttca
     5161 ggcccctcag tcctcacagt ctgttcatga tcataatcag ccataccaca tttgtagagg
     5221 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg
     5281 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     5341 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     5401 tcatcaatgt atcttaacgc gtaaattgta agcgtctcaa atatgtatcc gctcatgaga
     5461 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     5521 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     5581 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     5641 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     5701 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     5761 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     5821 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     5881 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     5941 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     6001 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     6061 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     6121 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     6181 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     6241 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     6301 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     6361 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6421 taatttaaaa ggatctaggt gaagatcctt tttgataatc
//
