LOCUS       sequence-387278-        5160 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid is designed for use in a procedure to synthesize 
            single-stranded DNA containing only the insert..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5160)
  AUTHORS   Strawn IK, Steiner PJ, Newton MS, Baumer ZT, Whitehead TA
  TITLE     A method for generating user-defined circular single-stranded DNA 
            from plasmid DNA using Golden Gate intramolecular ligation.
  JOURNAL   Biotechnol Bioeng. 2023 Oct;120(10):3057-3066. doi: 
            10.1002/bit.28471. Epub 2023 Jun 27.
  PUBMED    37366288
REFERENCE   2  (bases 1 to 5160)
  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"; doi: "10.1002/bit.28471";
            journalName: "Biotechnol Bioeng"; date: "2023-10"; volume: "120"; 
            issue: "10"; pages: "3057-3066"
COMMENT     Sequence Label: pIS001
FEATURES             Location/Qualifiers
     source          1..5160
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     718..737
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     802..821
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1035..1623
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1524..1543
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2037..2351
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    2045..2063
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2081..2099
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2116..2134
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2152..2170
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2188..2206
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2223..2241
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2259..2277
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2324..2348
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2381..2392
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3703..3744
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3766..3822
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(3823..3825,3826..3828,3829..4542)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3823..3825,3826..3828,3829..4539)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3868..3889)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4129..4148)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4476..4497
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4543..4767
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(join(5155..5160,1..810))
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(join(5155..5160,1..810))
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
ORIGIN
        1 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
       61 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
      121 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
      181 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
      241 cggtgagaat ggcaaaagct tatgcatttc tttccagact tgttcaacag gccagccatt
      301 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
      361 agcgaggcga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
      421 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
      481 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
      541 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
      601 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
      661 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
      721 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctgga
      781 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
      841 agacagtttt attgttcatg atgatatatt tttatcttgt gcaatgtaac atcagagatt
      901 ttgagacaca acgtggcttt gttgaataaa tcgaactttt gctgagttga aggatcagtc
      961 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     1021 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     1081 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     1141 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     1201 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     1261 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     1321 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     1381 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     1441 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     1501 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     1561 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     1621 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     1681 atgttctttc ctgcgttatc ccctgattct gtggataacc gtggtctctg ctgaggctgt
     1741 ctggtttcat gagtcatcag acttctaaga tcaggaaagg gaaaatgcca atgctctgtc
     1801 taggggttgg ataagccagt ataataaatg aaaatggggc taaaatgagt gttctaaaat
     1861 accttttgat aaggctgcag aaggagcggg agaaatggat atgaagtact gggctcttta
     1921 aaaatgatta aaattctgct tacatagtct aactcgcgac actgtaattt catactgtag
     1981 taaggatctc aagcaggaga gtataaaact cgggtgagca tgtctttaat ctacctgagt
     2041 ttactcccta tcagtgatag agaacgtatg tcgagtttac tccctatcag tgatagagaa
     2101 cgatgtcgag tttactccct atcagtgata gagaacgtat gtcgagttta ctccctatca
     2161 gtgatagaga acgtatgtcg agtttactcc ctatcagtga tagagaacgt atgtcgagtt
     2221 tatccctatc agtgatagag aacgtatgtc gagtttactc cctatcagtg atagagaacg
     2281 tatgtcgagg taggcgtgta cggtgggagg cctatataag cagagctcgt ttagtgaacc
     2341 gtcagatcgc ctattgtatc tgtttcaagg atacaaatac atcgagggtc gtttctgtta
     2401 cagattgcca ccatgaaaat gccattgacc tggtacagct ggttcctcct cagtgcctgg
     2461 atcctgaaca ctggggccga gatctccatc acccccgagc ctgcacaacc tgcagagggg
     2521 gacaatgtca cgctggtagt ccatgggctt tcaggggaac tgcttgccta caattggtac
     2581 gcagggccta cgctcagcct gaccttcctg gtggccagtt acattgtcag cactggtgat
     2641 gagacgcccg gaccagccca cacagggcgg gaagctgtcc gtcctgatgg cagccttgat
     2701 atccatggtg ccttacctgg gcacacaggc acctacatcc tgcagactct taacaggcag
     2761 tttcagacgg aggtgggcta cggacacatg caggtctatg agatcctggc ccctcccacg
     2821 gtcatggcca acgacactgc gctggtggag cgtagggata cccttcgtct cgtctgtagc
     2881 agccccagcc ccgcagaggt ccgctggttc ttcaatggtg acgctctgcc agttgccgtc
     2941 cgcttgggca tgtcccccga tggcaggatg ctgacccggc atggcgtccg cagggaggag
     3001 gcaggtgcct accagtgcga ggtgtggaac cccgtcagcg tcagccgcag cgagccactg
     3061 aacttgacgg tatacttcgg ccctgaacga gtagctatcc ttcaagactc taccacccgc
     3121 acgggctgca ctattaaagt ggacttcaac atgtccctca ccctgtggtg tgtggcgcgg
     3181 tcttgccctg agccagagta cgtgtgggcc ttcaatggaa aggccttgaa gaatggccag
     3241 gatcacctga acatcagcag tatgactgca gcccatgagg gcacgtacac gtgcatcgct
     3301 aagaactcca agacgctgct gtctggctct gcctcagtgg tagtcaagct gtccgcggca
     3361 gctgtagcca tgatgatcgt tcctgtaccg actaagccaa cggagggcca ggacgtgacc
     3421 ctgactgtcc agggctaccc caaggacctg ctagtctatg cctggtatcg tggacctgcc
     3481 tcggaaccca accgactgct cagccagctg ccgtcaggga actggatcgc tggcccggcg
     3541 cacacaggcc gggaggtggg cttcgctaat tgctcactgc tggtgcaaaa gctgaacctc
     3601 actgatgccg ggcgctacac actcaagacc gtcacactgc agggcaagac cgacactctg
     3661 gaggtggagc tacaggtggc ccccctggag ggcggaggaa gcggtaagcc tatccctaac
     3721 cctctcctcg gactcgattc tacgggaggc ggaagcggaa gcggagctac taacttcagc
     3781 ctgctgaagc aggctggaga cgtggaggag aaccctggac ctatggtgag caagggcgag
     3841 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     3901 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
     3961 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
     4021 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
     4081 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     4141 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     4201 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     4261 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
     4321 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     4381 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
     4441 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     4501 gccgccggga tcactctcgg catggacgag ctgtacaagt aactgtgcct tctagttgcc
     4561 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     4621 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     4681 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     4741 atgctgggga tgcggtgggc tctatggcga tggaaaatac tccgaggcgg atcacaagca
     4801 ataataacct gtagttttgc tgcataaaac cccagatgac tacctatcct cccattttcc
     4861 ttatttgccc ctattaaaaa acttcccgac aaaaccgaaa atctgtggga agtcttgtcc
     4921 ctccaatttt acacctgttc aattcccctg caggacaacg cccacacacc aggttagcct
     4981 ttaagcctgc ccagaagact cccgcccatc ttctagaaag actggagttg cagatcacga
     5041 gggaagaggg ggaagggatt ctcccaggcc cagggcggtc ctcagaagcc aggaggcagc
     5101 agagaactcc cagaagctgt gagaccgcgt ttacaaccaa ttaaccaatt ctgattagaa
//
