LOCUS       sequence-333564-        5739 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression for structure determination; may not be full 
            ORF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5739)
  TITLE     SGC+year
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 5739)
  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: 
            "unpublished"
COMMENT     Sequence Label: ASH1L
FEATURES             Location/Qualifiers
     source          1..5739
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..78)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     284..303
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     387..406
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        387..405
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    406..430
                     /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)."
     RBS             445..467
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             478..495
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             520..540
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             990..1007
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1060..1078)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1074..1121
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1158..1613
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1245..1264)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1455..1476
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1705..2520)
                     /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(1705..2520)
                     /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     2428..2447
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2512..2531
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2642..3230
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3131..3150
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3384..3401
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3416..3555
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3481..3500)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3641..3663
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3657..3848)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4657..5739)
                     /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     5701..5720
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
       61 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
      121 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
      181 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
      241 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
      301 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
      361 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
      421 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat acatatgcac
      481 catcatcatc atcattcttc tggtgtagat ctgggtaccg agaacctgta cttccaatcc
      541 atggaagtgg cgcgtgcggc gcgtctggcc cagattttta aagaaatttg cgatggcatt
      601 attagctata aagatagcag ccgtcaggcg ctggccgccc cgctgctgaa cctgccgccg
      661 aaaaaaaaaa atgccgatta ttatgaaaaa attagcgatc cgctggatct gattaccatc
      721 gaaaaacaga ttctgaccgg ttattataaa accgtggaag cgtttgatgc cgatatgctg
      781 aaagtgtttc gtaatgccga aaaatattat ggtcgcaaaa gcccggttgg ccgcgatgtt
      841 tgtcgcctgc gtaaagcgta ttataacgcc cgtcatgaag cgagcgccca gattgatgaa
      901 attgtgggcg aaaccgccag cgaatgacag taaaggtgga tacggatccg aattcgagct
      961 ccgtcgacaa gcttgcggcc gcactcgagc accaccacca ccaccactga gatccggctg
     1021 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
     1081 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     1141 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     1201 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     1261 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     1321 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     1381 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     1441 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     1501 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     1561 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     1621 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     1681 atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa
     1741 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     1801 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
     1861 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     1921 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc
     1981 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     2041 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa
     2101 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     2161 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     2221 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     2281 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     2341 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     2401 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     2461 atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat
     2521 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc
     2581 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     2641 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     2701 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     2761 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     2821 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     2881 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     2941 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     3001 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     3061 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     3121 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     3181 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     3241 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     3301 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     3361 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     3421 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     3481 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     3541 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     3601 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     3661 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     3721 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     3781 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     3841 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     3901 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     3961 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     4021 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     4081 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     4141 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     4201 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     4261 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc
     4321 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     4381 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     4441 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     4501 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     4561 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     4621 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4681 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4741 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     4801 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     4861 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     4921 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     4981 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     5041 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     5101 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     5161 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     5221 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     5281 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     5341 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     5401 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     5461 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     5521 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     5581 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     5641 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     5701 ggcatactct gcgacatcgt ataacgttac tggtttcac
//
