LOCUS       sequence-309017-        6813 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses API5 in E.coli cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6813)
  AUTHORS   Bong SM, Bae SH, Song B, Gwak H, Yang SW, Kim S, Nam S, Rajalingam 
            K, Oh SJ, Kim TW, Park S, Jang H, Lee BI
  TITLE     Regulation of mRNA export through API5 and nuclear FGF2 interaction.
  JOURNAL   Nucleic Acids Res. 2020 Jun 19;48(11):6340-6352. doi: 
            10.1093/nar/gkaa335.
  PUBMED    32383752
REFERENCE   2  (bases 1 to 6813)
  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"; journalName: 
            "Nucleic Acids Res"; date: "2020-06-19- 19"; volume: "48"; issue: 
            "11"; pages: "6340-6352"
COMMENT     Sequence Label: pET28b-API5
FEATURES             Location/Qualifiers
     source          1..6813
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1585..1776
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1770..1792)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1878..2017
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1933..1952
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2032..2049)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2203..2791)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2283..2302)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2902..2921)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2913..3728
                     /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             2913..3728
                     /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"
     primer_bind     complement(2986..3005)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3820..4275)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3957..3978)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4169..4188
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4312..4359)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4355..4373
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4426..4443)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(5977..5994)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(6004..6021)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             6040..6062
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    6077..6101
                     /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)."
     primer_bind     complement(6101..6120)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6102..6120)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6204..6223)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6429..6506
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(6507..6813,1..776)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(6526..6545)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga
       61 tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg
      121 ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt attttctccc
      181 atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg
      241 cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata
      301 aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca
      361 tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc
      421 tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc
      481 gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata
      541 tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc
      601 gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac
      661 tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg
      721 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
      781 aacgcaatta atgtaagtta gctcactcat taggcaccgg gatctcgacc gatgcccttg
      841 agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca
      901 cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc gctctgggtc
      961 attttcggcg aggaccgctt tcgctggagc gcgacgatga tcggcctgtc gcttgcggta
     1021 ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc
     1081 ggcgagaagc aggccattat cgccggcatg gcggccccac gggtgcgcat gatcgtgctc
     1141 ctgtcgttga ggacccggct aggctggcgg ggttgcctta ctggttagca gaatgaatca
     1201 ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca
     1261 acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc
     1321 tgcaccatta tgttccggat ctgcatcgca ggatgctgct ggctaccctg tggaacacct
     1381 acatctgtat taacgaagcg ctggcattga ccctgagtga tttttctctg gtcccgccgc
     1441 atccataccg ccagttgttt accctcacaa cgttccagta accgggcatg ttcatcatca
     1501 gtaacccgta tcgtgagcat cctctctcgt ttcatcggta tcattacccc catgaacaga
     1561 aatccccctt acacggaggc atcagtgacc aaacaggaaa aaaccgccct taacatggcc
     1621 cgctttatca gaagccagac attaacgctt ctggagaaac tcaacgagct ggacgcggat
     1681 gaacaggcag acatctgtga atcgcttcac gaccacgctg atgagcttta ccgcagctgc
     1741 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     1801 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     1861 gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact
     1921 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccattgc ggtgtgaaat
     1981 accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac
     2041 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     2101 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     2161 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     2221 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     2281 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     2341 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     2401 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     2461 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     2521 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     2581 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     2641 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     2701 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     2761 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     2821 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgaaca ataaaactgt
     2881 ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcaacgg gaaacgtctt
     2941 gctctaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc
     3001 gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc
     3061 cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg
     3121 tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta
     3181 ctcctgatga tgcatggtta ctcaccactg cgatccccgg gaaaacagca ttccaggtat
     3241 tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc
     3301 ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg
     3361 ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc
     3421 gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg ccattctcac
     3481 cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga
     3541 aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg
     3601 ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa
     3661 aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt
     3721 ttttctaaga attaattcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     3781 ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt
     3841 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     3901 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     3961 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     4021 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     4081 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     4141 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     4201 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     4261 cgctacaggg cgcgtcccat tcgccaatcc ggatatagtt cctcctttca gcaaaaaacc
     4321 cctcaagacc cgtttagagg ccccaagggg ttatgctagt tattgctcag cggtggcagc
     4381 agccaactca gcttcctttc gggctttgtt agcagccgga tctcagtggt ggtggtggtg
     4441 gtgctcgagt gcggccgctt acttcccctg aaggctcctc tcataattaa agttgcccaa
     4501 attgctgcta tatttcccac tgggagggtt ataaatctgc ctggcatctc tttttggtcc
     4561 tgctgaagat ttcttgggtg gtgaacctga agttgtatct tcactggctc tcttttgccc
     4621 aatctcaacc ttttgtacag gtttccagga tagtgttact gtgctcttat aagaaggagg
     4681 aatgtggaag agatccttga ttaaaacatt gatattgttt gttattttca atgcaacgac
     4741 tttaatcttg ttctcttctg tttttaaggc ctcacccgtt ttaccctgga gagctaagcg
     4801 aagttgtctg atataaactt gcaggccccg tgcaaagtac tgcagcctga ttttgaaatc
     4861 tttgagcttt tctgcattca gtttggctgt taagaaatct ggaagttttc ggcccaactg
     4921 gtgaaaactg tacaacaaac attccacata actgaactgt agcttgggtt cttcattacc
     4981 agcattctct ccattttctg cctcttctgg agggaggggc atgtattcca ataacttatc
     5041 aaatagtttc cttaaatttg tttctagttt ttccatgtca ccacaaaatg aactcatctc
     5101 cgccaacaat ttcaatacct ccaactgtat atcaagacct tccactgggg tagtcaaggt
     5161 accgaggtta gggagaacct gctcacagaa atatgtcaca aaccttgtgg aatggacatt
     5221 tttagagaag aggggtactg cctgccgagt gcactgtaag agcctgtcca cacagtcagg
     5281 atccgaggga ttgaaggtct gttctaggtc ggcctgttca gccaccaact ctacaagttg
     5341 ctgtcttcca ctcactgtct gtaagctttt taacccagac agtatcttca taaatagaac
     5401 aaattcttca ccagtcacat cttctaggac ctttttggat tcagttagta taagctcttc
     5461 cacttccttt gttaagactt catctggtaa agtcttaagt tttgtagaaa ggaatttaat
     5521 tgctcgttct ctaacaatgt cctctccttg aagtatttgg ctgaacaacc cacctaaagt
     5581 cccttttgca tccattttaa atatacttaa tagggcattg ttcactaggt taaattctgc
     5641 agagtcatct gtctgcaaaa gttgcgttag tatatctgcc actcgaggaa gattttctcc
     5701 agtggcaaat tgaggcagtt ctttaattgc ttgacgtcga atagatacat cttcatcctc
     5761 acagaggtct aactgtgcat tgatagcaga atcagccaat tctggaaaat gcttaaagaa
     5821 tttcggaata aattgagctg ctaatcgctt ttccttagta ccacctttca caccatccaa
     5881 tatcacttga taggcatctt tatgctggcc cacttgctcc gtggcatcgg ccaggatgcc
     5941 ataattgcgg taaagctcct ctactgtcgg catatggctg ccgcgcggca ccaggccgct
     6001 gctgtgatga tgatgatgat ggctgctgcc catggtatat ctccttctta aagttaaaca
     6061 aaattatttc tagaggggaa ttgttatccg ctcacaattc ccctatagtg agtcgtatta
     6121 atttcgcggg atcgagatct cgatcctcta cgccggacgc atcgtggccg gcatcaccgg
     6181 cgccacaggt gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc
     6241 tcgccacttc gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc
     6301 cgggggactg ttgggcgcca tctccttgca tgcaccattc cttgcggcgg cggtgctcaa
     6361 cggcctcaac ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgtcg
     6421 agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg
     6481 aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt
     6541 atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg
     6601 cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg
     6661 tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg
     6721 ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg
     6781 ccagcgtggt ggtgtcgatg gtagaacgaa gcg
//
