LOCUS       sequence-153571-        6258 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human alpha A-crystallin in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6258)
  TITLE     Larry David lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6258)
  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            "Unpublished"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: pE-SUMO CRYAA
FEATURES             Location/Qualifiers
     source          1..6258
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..40
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    40..64
                     /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             79..101
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             114..131
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             144..431
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated and modified)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /note="modified to prevent cleavage by eukaryotic SUMO 
                     proteases"
                     /translation="DSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRR
                     LMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG"
     CDS             1006..1023
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1076..1094)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1090..1137
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1174..1629
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1261..1280)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1471..1492
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1656..1760
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1761..1829,1830..2621)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1979..1998)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2792..3380
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3281..3300
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3534..3551
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3566..3708
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3634..3653)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3794..3816
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3810..4001)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4810..5892)
                     /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     5854..5873
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5893..5970)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6176..6195
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
       61 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg ggtcatcacc
      121 atcatcatca cgggtccctg caggactcag aagtcaatca agaagctaag ccagaggtca
      181 agccagaagt caagcctgag actcacatca atttaaaggt gtccgatgga tcttcagaga
      241 tcttcttcaa gatcaaaaag accactcctt taagaaggct gatggaagcg ttcgctaaaa
      301 gacagggtaa ggaaatggac tccttaagat tcttgtacga cggtattaga attcaagctg
      361 atcaggcccc tgaagatttg gacatggagg ataacgatat tattgaggct caccgcgaac
      421 agattggagg tatggatgtg accatccagc acccctggtt caagcgcacc ctggggccct
      481 tctaccccag ccggctgttc gaccagtttt tcggcgaggg cctttttgag tatgacctgc
      541 tgcccttcct gtcgtccacc atcagcccct actaccgcca gtccctcttc cgcaccgtgc
      601 tggactccgg catctctgag gttcgatccg accgggacaa gttcgtcatc ttcctcgatg
      661 tgaagcactt ctccccggag gacctcaccg tgaaggtgca ggacgacttt gtggagatcc
      721 acggaaagca caacgagcgc caggacgacc acggctacat ttcccgtgag ttccaccgcc
      781 gctaccgcct gccgtccaac gtggaccagt cggccctctc ttgctccctg tctgccgatg
      841 gcatgctgac cttctgtggc cccaagatcc agactggcct ggatgccacc cacgccgagc
      901 gagccatccc cgtgtcgcgg gaggagaagc ccacctcggc tccctcgtcc taatctagag
      961 gatccgaatt cgagctccgt cgacaagctt gcggccgcac tcgagcacca ccaccaccac
     1021 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     1081 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     1141 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     1201 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     1261 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     1321 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     1381 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     1441 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     1501 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1561 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     1621 ttacaatttc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1681 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     1741 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     1801 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     1861 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     1921 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     1981 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     2041 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     2101 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     2161 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     2221 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     2281 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
     2341 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     2401 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     2461 gagccggtga gcgtggttct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     2521 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     2581 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     2641 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     2701 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     2761 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2821 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     2881 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     2941 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     3001 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3061 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3121 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3181 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3241 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     3301 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     3361 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     3421 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     3481 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     3541 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     3601 gtatttcaca ccgcatatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     3661 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     3721 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     3781 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     3841 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     3901 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     3961 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     4021 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     4081 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     4141 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     4201 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     4261 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     4321 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     4381 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     4441 gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc
     4501 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     4561 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     4621 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     4681 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     4741 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     4801 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4861 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     4921 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     4981 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     5041 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc
     5101 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     5161 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     5221 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     5281 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     5341 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     5401 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     5461 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     5521 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     5581 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     5641 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     5701 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     5761 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     5821 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     5881 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     5941 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     6001 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     6061 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     6121 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     6181 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     6241 gtccggcgta gaggatcg
//
