LOCUS       sequence-492596-        6462 bp    DNA     circular SYN 11-NOV-2025
DEFINITION  Expression of eeBxb1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6462)
  AUTHORS   Migliori V, Bruntraeger MB, Guylev IS, Burgold T, Lichou F, Trinh 
            AL, Washer SJ, Jones CP, Trynka G, Bassett AR
  TITLE     ONE-STEP tagging: a versatile method for rapid site-specific 
            integration by simultaneous reagent delivery
  JOURNAL   bioRxiv 2024.08.29.610246
REFERENCE   2  (bases 1 to 6462)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 11, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2024.08.29.610246"
COMMENT     Sequence Label: EF1a_eeBxb1_Int_2x_NPL_c-term_NLS_pTwist
FEATURES             Location/Qualifiers
     source          1..6462
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(1..589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(81..100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             join(1335..1403,1404..2195)
                     /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(1553..1572)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      2208..2344
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2496..2567
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(2572..2618)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    2636..2684
                     /note="synthetic polyadenylation signal"
     promoter        2721..3899
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2952..3890
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3847..3867
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             5500..5547
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5557..5604
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    5673..5794
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5684..5703)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5738..5757
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      5909..5955
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      5985..6121
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6166..6237
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
ORIGIN
        1 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
       61 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      121 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      181 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      241 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      301 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      361 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      421 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
      481 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      541 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
      601 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
      661 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
      721 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
      781 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcattta aatttccgaa
      841 ctctccaagg ccctcgtcgg aaaatcttca aacctttcgt ccgatccatc ttgcaggcta
      901 cctctcgaac gaactatcgc aagtctcttg gccggccttg cgccttggct attgcttggc
      961 agcgcctatc gccaggtatt actccaatcc cgaatatccg agatcgggat cacccgagag
     1021 aagttcaacc tacatcctca atcccgatct atccgagatc cgaggaatat cgaaatcggg
     1081 gcgcgcctgg tgtaccgaga acgatcctct cagtgcgagt ctcgacgatc catatcgttg
     1141 cttggcagtc agccagtcgg aatccagctt gggacccagg aagtccaatc gtcagatatt
     1201 gtactcaagc ctggtcacgg cagcgtaccg atctgtttaa acctagatat tgatagtctg
     1261 atcggtcaac gtataatcga gtcctagctt ttgcaaacat ctatcaagag acaggatcag
     1321 caggaggctt tcgcatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     1381 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     1441 atcagttggg tgcgcgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     1501 agagttttcg ccccgaagaa cgctttccaa tgatgagcac ttttaaagtt ctgctatgtg
     1561 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     1621 ctcagaatga cttggttgag tattcaccag tcacagaaaa gcatcttacg gatggcatga
     1681 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     1741 ttctgacaac gattggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     1801 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     1861 gtgacaccac gatgcctgta gcaatggcaa caaccttgcg taaactatta actggcgaac
     1921 tacttactct agcttcccgg caacagttga tagactggat ggaggcggat aaagttgcag
     1981 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     2041 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     2101 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     2161 ctgagatagg tgcctcactg attaagcatt ggtaaccgat tctaggtgca ttggcgcaga
     2221 aaaaaatgcc tgatgcgacg ctgcgcgtct tatactccca catatgccag attcagcaac
     2281 ggatacggct tccccaactt gcccacttcc atacgtgtcc tccttaccag aaatttatcc
     2341 ttaagatccc gaatcgttta aactcgactc tggctctatc gaatctccgt cgtttcgagc
     2401 ttacgcgaac agccgtggcg ctcatttgct cgtcgggcat cgaatctcgt cagctatcgt
     2461 cagcttacct ttttggcagc gatcgcggct cccgacatct tggaccatta gctccacagg
     2521 tatcttcttc cctctagtgg tcataacagc agcttcagct acctctcaat tcaaaaaacc
     2581 cctcaagacc cgtttagagg ccccaagggg ttatgctatc aatcgttgcg ttacacacac
     2641 aaaaaaccaa cacacatcca tcttcgatgg atagcgattt tattatctaa ctgctgatcg
     2701 agtgtagcca gatctcgtga ggctccggtg cccgtcagtg ggcagagcgc acatcgccca
     2761 cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc
     2821 gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg
     2881 gagaaccgta tataagtgca ctagtcgccg tgaacgttct ttttcgcaac gggtttgccg
     2941 ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg
     3001 gcccttgcgt gccttgaatt acttccacct ggctgcagta cgtgattctt gatcccgagc
     3061 ttcgggttgg aagtgggtgg gagagttcgt ggccttgcgc ttaaggagcc ccttcgcctc
     3121 gtgcttgagt tgtggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc
     3181 ttcgcgcctg tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg
     3241 ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc gggccaagat cagcacactg
     3301 gtatttcggt ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt
     3361 cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg
     3421 cccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa
     3481 ggctggcccg gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctg
     3541 cagggagcac aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac
     3601 aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg
     3661 cgccgtccag gcacctcgat tagttctcca gcttttggag tacgtcgtct ttaggttggg
     3721 gggaggggtt ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc
     3781 cagcttggca cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt
     3841 tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcgtgaa
     3901 aactacccct aaaagccaaa agatctttgt cgatcctacc atccactcga cacacccgcc
     3961 agcggccgct gccaagcttc cgagctctcg aattccacca tgagagccct ggtcgtgatt
     4021 agactgagca gagtgacaga cgctactact tcccctgaga gacagctgga gtcatgccag
     4081 cagctgtgcg cacagagagg atgggacgtg gtcggagtgg cagaggacct ggacgtgagc
     4141 ggcgctgtcg atccatttga cagaaagcgg agacccaacc tggcaaggtg gctggctttc
     4201 gaggaacagc cctttgatgc gatcgtcgcc tacagagtgg acaggctgac acgctctatt
     4261 cgacatctgc agcagctggt gcattgggcc gaggaccaca agaaactggt ggtcagtgca
     4321 actgaagccc acttcgatac cacaactcct tttgccgctg tggtcatcgc actgatgggc
     4381 accgtggccc agatggagct ggaagctatc aaggagcgaa accggagtgc agcccatttc
     4441 aatattcggg ccgggaaata cagaggatca ctgccccctt ggggctatct gcctacccgg
     4501 gtggatgggg agtggagact ggtgccagac cccgtccaga gagagaggat tctggaagtg
     4561 taccaccggg tggtcgataa ccacgaacca ctgcatctgg tcgcccacga cctgaatagg
     4621 cgcggcgtgc tgagcccaaa agattatttt gctcagctgc agggaaggga gccacaggga
     4681 cgaaaatggt ccgctaccgc cctgaagcgg agcatgatca gtgaggctat gctgggctat
     4741 gcaactctga atgggaaaac cgtccgggac gatgacggag cccctctggt gcgggctgag
     4801 cctattctga cacgcgagca gctggaagct ctgcgggcag aactggtgaa gaccagtaga
     4861 gccaaacctg ccgtgagcac accaagcctg ctgctgaggg tgctgttctg cgccgtctgt
     4921 ggggagccag catacaagtt tgccggcggg ggaagaaaac atccccgcta tagatgccgg
     4981 agcatgggat tccctaagca ctgtggaaac ggcactgtgg ctatggctga gtgggacgcc
     5041 ttttgcgagg aacaggtgct ggatctgctg ggagacgccg agaggctgga aaaagtgtgg
     5101 gtcgctggca gcgactccgc tatcgagctg gcagaagtca atgccgagct ggtggatctg
     5161 acctccctga tcggatctcc tgcatacagg gcaggctccc cacagcgaga agctctggac
     5221 gcacgaattg ctgcactggc agctcgacag gaggaactgg agggactgga agcacgacct
     5281 tctggatggg agtggcgaga aacaggccag cggtttgggg attggtggcg cgagcaggac
     5341 acagcagcca agaacacttg gctgagaagc atgaatgtca ggctgacttt cgatgtgcgc
     5401 ggcgggctga cccgaaccat tgattttggc gacctgcagg agtatgaaca gcacctgagg
     5461 ctggggagtg tggtggagag actgcatact ggaatgagca agagaccagc ggcgacgaag
     5521 aaagcgggcc aggcgaaaaa aaagaaggga agcggcaaaa ggccggcggc cacgaaaaag
     5581 gccggccagg caaaaaagaa aaagtgagga tccgcaggcc tctgctagct tgactgactg
     5641 agatacagcg taccttcagc tcacagacat gataagatac attgatgagt ttggacaaac
     5701 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     5761 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     5821 gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg
     5881 tggtattggc ccatctctat cggtatcgta gcataacccc ttggggcctc taaacgggtc
     5941 ttgaggggtt ttttgtgccc ctcgggccgg attgctatct accggcattg gcgcagaaaa
     6001 aaatgcctga tgcgacgctg cgcgtcttat actcccacat atgccagatt cagcaacgga
     6061 tacggcttcc ccaacttgcc cacttccata cgtgtcctcc ttaccagaaa tttatcctta
     6121 aggtcgtcag ctatcctgca ggcgatctct cgatttcgat caagacattc ctttaatggt
     6181 cttttctgga caccactagg ggtcagaagt agttcatcaa actttcttcc ctccctaatc
     6241 tcattggtta ccttgggcta tcgaaactta attaaccagt caagtcagct acttggcgag
     6301 atcgacttgt ctgggtttcg actacgctca gaattgcgtc agtcaagttc gatctggtcc
     6361 ttgctattgc acccgttctc cgattacgag tttcatttaa atcatgtgag caaaaggcca
     6421 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tt
//
