LOCUS       sequence-199971-        5988 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 5988)
  TITLE     SGC+year
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 5988)
  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            "unpublished"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: SPSB1
FEATURES             Location/Qualifiers
     source          1..5988
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1319..1510
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1504..1526)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1612..1751
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1667..1686
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1766..1783)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1937..2525)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2017..2036)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2636..2655)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2647..3462
                     /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             2647..3462
                     /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(2720..2739)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3554..4009)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3691..3712)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3903..3922
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4046..4093)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4089..4107
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4160..4177)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4876..4896)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             complement(4921..4938)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             4949..4971
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4986..5010
                     /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(5010..5029)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5011..5029)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5113..5132)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5338..5415
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5416..5988,1..510)
                     /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(5435..5454)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag
       61 ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa
      121 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg
      181 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg
      241 ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag
      301 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag
      361 gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg
      421 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
      481 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac
      541 tcattaggca ccgggatctc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt
      601 ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca
      661 actcgtagga caggtgccgg cagcgctctg ggtcattttc ggcgaggacc gctttcgctg
      721 gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca
      781 agccttcgtc actggtcccg ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg
      841 catggcggcc ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg
      901 gcggggttgc cttactggtt agcagaatga atcaccgata cgcgagcgaa cgtgaagcga
      961 ctgctgctgc aaaacgtctg cgacctgagc aacaacatga atggtcttcg gtttccgtgt
     1021 ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc attatgttcc ggatctgcat
     1081 cgcaggatgc tgctggctac cctgtggaac acctacatct gtattaacga agcgctggca
     1141 ttgaccctga gtgatttttc tctggtcccg ccgcatccat accgccagtt gtttaccctc
     1201 acaacgttcc agtaaccggg catgttcatc atcagtaacc cgtatcgtga gcatcctctc
     1261 tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg aggcatcagt
     1321 gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac
     1381 gcttctggag aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct
     1441 tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga
     1501 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     1561 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat
     1621 gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag
     1681 attgtactga gagtgcacca ttgcggtgtg aaataccgca cagatgcgta aggagaaaat
     1741 accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     1801 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     1861 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     1921 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     1981 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     2041 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     2101 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     2161 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     2221 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2281 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     2341 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     2401 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     2461 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     2521 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     2581 gttaagggat tttggtcatg aacaataaaa ctgtctgctt acataaacag taatacaagg
     2641 ggtgttatga gccatattca acgggaaacg tcttgctcta ggccgcgatt aaattccaac
     2701 atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg
     2761 acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa
     2821 ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt
     2881 atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc
     2941 actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa
     3001 aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat
     3061 tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac
     3121 ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc
     3181 tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat
     3241 ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga
     3301 cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag
     3361 ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg
     3421 aataaattgc agtttcattt gatgctcgat gagtttttct aagaattaat tcatgagcgg
     3481 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     3541 aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     3601 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     3661 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     3721 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     3781 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     3841 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     3901 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     3961 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
     4021 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
     4081 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
     4141 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
     4201 cgacggagct cgaattcgga tccgtatcca cctttactgt cactcgggat cgagtccgtt
     4261 caagtagcgc attcggatct cacagtggcc ccagacggca ctcactacag gatacagttt
     4321 tttgcccttg agtccccgaa aagccactcc catgtactgt ccatccacaa tgaagctcag
     4381 agtcccgtcg tccatgtcca gggctaccag gaaggagtca gggacaatga atgtctcatc
     4441 tggttccaga aaggctgggt atgttttgct tggctggttc ttgccatcgt ggtagagccg
     4501 gttgcgcccc aagtcccagc cccaggactc gtggttattc cccacgaggg ttgtgtaccc
     4561 gacagagtgc aggggggcgt ctgccgtcgc cacccccacc acggcgtgtg tgccccgctg
     4621 tctcatggcc cacgtgatct gccacacgtg cagcccacgg gtatacccga ctttgcccct
     4681 gatagcgtcc gtgctctggg ccaccggatg ccggtgaaag atgagcttgt cgtcctcctt
     4741 cacaaagaca ttgagcgatc ggtcgttgtt gttccatgaa tgcagcagct ggacatcata
     4801 ggacacaggg ggcatgtcca gtagcagatc cagccgggtg ggcttgcagt aatccagacc
     4861 ctggagctcc tgcatggatt ggaagtacag gttctcggta cccagatcta caccagaaga
     4921 atgatgatga tgatggtgca tatgtatatc tccttcttaa agttaaacaa aattatttct
     4981 agaggggaat tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga
     5041 tcgagatctc gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg
     5101 cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg
     5161 ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt
     5221 tgggcgccat ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc
     5281 tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga gatcccggac
     5341 accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa
     5401 ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc
     5461 tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg
     5521 gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa
     5581 ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg
     5641 ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg
     5701 gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc
     5761 gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct
     5821 gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc
     5881 atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc
     5941 gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttct
//
