LOCUS       sequence-162056-        7794 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IPTG inducible, cLac143 (Markley 2015), LDH V39R with Gmr in glpK.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7794)
  AUTHORS   Gordon GC, Korosh TC, Cameron JC, Markley AL, Begemann MB, Pfleger 
            BF
  TITLE     CRISPR interference as a titratable, trans-acting regulatory tool 
            for metabolic engineering in the cyanobacterium Synechococcus sp. 
            strain PCC 7002.
  JOURNAL   Metab Eng. 2016 Jul 29. pii: S1096-7176(16)30062-3. doi: 
            10.1016/j.ymben.2016.07.007.
  PUBMED    27481676
REFERENCE   2  (bases 1 to 7794)
  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: "Metab Eng. 
            2016 Jul 29. pii: S1096-7176(16)30062-3. doi: 
            10.1016/j.ymben.2016.07.007."
COMMENT     Sequence Label: pCas61
FEATURES             Location/Qualifiers
     source          1..7794
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(64..83)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     274..295
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        510..614
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(615..683,684..1475)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(833..852)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1646..2234
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2135..2154
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2388..2405
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3149..3168
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     protein_bind    3253..3269
                     /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)."
     CDS             3318..3347
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             3360..3371
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4410..4428)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4424..4471
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        4569..4603
                     /label=BBa_J23110
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.33"
     CDS             4633..5715
                     /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
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDFSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4652..4671)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5984..5989,5990..6006,6007..6012)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             6201..6734
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEVVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     primer_bind     complement(7588..7607)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7589..7607)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7614..7631)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7614..7630)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7623..7645)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      join(7771..7794,1..432)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
       61 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
      121 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
      181 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
      241 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
      301 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
      361 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
      421 cgcttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
      481 catcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
      541 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
      601 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
      661 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
      721 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
      781 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
      841 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
      901 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
      961 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     1021 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     1081 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     1141 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     1201 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     1261 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     1321 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     1381 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     1441 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     1501 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     1561 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     1621 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     1681 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     1741 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     1801 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     1861 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     1921 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     1981 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2041 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     2101 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     2161 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     2221 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     2281 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     2341 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     2401 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     2461 attaatgcag ctggcacgac aggtttcccg actggaagcg attggctatg atctaccaaa
     2521 gctggcgatt tttctgcaaa ccgaattaga accacaattt gattttttaa agccgattac
     2581 cattgccaac ctgaaggaaa tagcgatcgc ctttatggat gatggccacg aggcttgtcg
     2641 ggtctcccat tattgtggat cggtaccgga tgagcgggcg agctttaatt tacgcttgcg
     2701 gcaatatcgc caggcccagc cttggctgcg aaatcatctc gatcctgcaa ggggcgatcg
     2761 cctgcaccat tggtcggatc accaacgcac cattttctac ggcaggcgca ccaatcccga
     2821 cacccagcag cggcttgtgt tagtggcgca catggccggg gctccgaaga ccgttgagat
     2881 tggcaaatgg ctcgccctgg atttggatcg ttggcagttg gcgatcgcca caccgacttt
     2941 gaagatcaac accatctatg acttagccca aattcacttg cacaatggcg aaggttttct
     3001 gttatctgaa attcctccct aaatgatgtc ttttgagcct aaaaacacac ttttttgacc
     3061 taatttaacc catttaaaaa actttattta ataatgacca tggcccatca aaaatacatt
     3121 cttgcgttag acctcggtac cacaaacgaa ttgtgagcgc tcacaattcg gaattcttaa
     3181 caaaaaagca ggaataaaat taacaagatg taattgacat aagtcccatc accgttgtat
     3241 aaatgtgtgg aattgtgagc ggataacaat ttcacacacc aactcataaa gtcaagtagg
     3301 agattaattc catgggccat catcatcatc atcatcatca tcatcacagc agcggccata
     3361 tcgaaggtcg tcatatgatg aacaaacatg taaataaagt agctttaatc ggagcgggtt
     3421 ttgttggaag cagttatgca tttgcgttaa ttaaccaagg aatcacagat gagcttgtgg
     3481 tcattgatcg taataaagaa aaagcaatgg gcgatgtgat ggatttaaac cacggaaagg
     3541 cgtttgcgcc acaaccggtc aaaacatctt acggaacata tgaagactgc aaggatgctg
     3601 atattgtctg catttgcgcc ggagcaaacc aaaaacctgg tgagacacgc cttgaattag
     3661 tagaaaagaa cttgaagatt ttcaaaggca tcgttagtga agtcatggcg agcggatttg
     3721 acggcatttt cttagtcgcg acaaatccgg ttgatatcct gacttacgca acatggaaat
     3781 tcagcggcct gccaaaagag cgggtgattg gaagcggcac aacacttgat tctgcgagat
     3841 tccgtttcat gctgagcgaa tactttggcg cagcgcctca aaacgtacac gcgcatatta
     3901 tcggagagca cggcgacaca gagcttcctg tttggagcca cgcgaatgtc ggcggtgtgc
     3961 cggtcagtga actcgttgag aaaaacgatg cgtacaaaca agaggagctg gaccaaattg
     4021 tagatgatgt gaaaaacgca gcttaccata tcattgagaa aaaaggcgcg acttattatg
     4081 gggttgcgat gagtcttgct cgcattacaa aagccattct tcataatgaa aacagcatat
     4141 taactgtcag cacatatttg gacgggcaat acggtgcaga tgacgtgtac atcggtgtgc
     4201 cggctgtcgt gaatcgcgga gggatcgcag gtatcactga gctgaactta aatgagaaag
     4261 aaaaagaaca gttccttcac agcgccggcg tccttaaaaa cattttaaaa cctcattttg
     4321 cagaacaaaa agtcaactaa cgagggcggt gctttggcag gatccggctg ctaacaaagc
     4381 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     4441 ggcctctaaa cgggtcttga cgggtttttt gtctagatca acggcctcaa cctactactg
     4501 ggctgcttcc taatgcagga gtcgcataag ggagagcgtc gagatcccgg acaccatcga
     4561 atggcgcatt tacagctagc tcagtcctag gtacaatgct agccggagga gggtcaattc
     4621 atggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
     4681 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
     4741 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
     4801 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
     4861 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
     4921 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
     4981 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
     5041 gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc
     5101 aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca
     5161 ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg
     5221 cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg
     5281 gaaggcgact ttagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc
     5341 atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc
     5401 attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc
     5461 gaagacagct catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg
     5521 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat
     5581 cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc
     5641 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     5701 gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcaccggg
     5761 atctcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt tctcgacgca
     5821 tgcataaaaa ctgttgtaat tcattaagca ttctgccgac atggaagcca tcacaaacgg
     5881 catgatgaac ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta taatatttgc
     5941 ccatggacgc acaccgtgga aacggatgaa ggcacgaacc cagttgacat aagcctgttc
     6001 ggttcgtaaa ctgtaatgca agtagcgtat gcgctcacgc aactggtcca gaaccttgac
     6061 cgaacgcagc ggtggtaacg gcgcagtggc ggttttcatg gcttgttatg actgtttttt
     6121 tgtacagtct atgcctcggg catccaagca gcaagcgcgt tacgccgtgg gtcgatgttt
     6181 gatgttatgg agcagcaacg atgttacgca gcagcaacga tgttacgcag cagggcagtc
     6241 gccctaaaac aaagttaggt ggctcaagta tgggcatcat tcgcacatgt aggctcggcc
     6301 ctgaccaagt caaatccatg cgggctgctc ttgatctttt cggtcgtgag ttcggagacg
     6361 tagccaccta ctcccaacat cagccggact ccgattacct cgggaacttg ctccgtagta
     6421 agacattcat cgcgcttgct gccttcgacc aagaagtggt tgttggcgct ctcgcggctt
     6481 acgttctgcc caagtttgag cagccgcgta gtgagatcta tatctatgat ctcgcagtct
     6541 ccggagagca ccggaggcag ggcattgcca ccgcgctcat caatctcctc aagcatgagg
     6601 ccaacgcgct tggtgcttat gtgatctacg tgcaagcaga ttacggtgac gatcccgcag
     6661 tggctctcta tacaaagttg ggcatacggg aagaagtgat gcactttgat atcgacccaa
     6721 gtaccgccac ctaacaattc gttcaagccg agatcggctt cccggccgcg gagttgttcg
     6781 gtaaattgtc acaacgccgc aggtggcact tttcggggaa atgtgcgcgc ccgcgttcct
     6841 gctggcgctg ggcctgtttc tggcgctgga cttcccgctg ttccgtcagc agcttttcgc
     6901 ccacggcctt gatgatcgcg gcggccttgg cctgcatatc ccgattcaac ggccccaggg
     6961 cgtccagaac gggcttcagg cgctcccgaa ggtggatccc cccctcttgc ctacagcatc
     7021 tcccccaggg gagaattctt cctgtttcaa ctccctctaa cgtaaaccca ttgaatttaa
     7081 aaaagacttt atgactgctt tactgctcca tgaccaacat tattcccttg atcatgaagc
     7141 ctttctctca accctcagca acacagaaaa tttactcatt attcaagatc tagatggcgt
     7201 ttgcatgggg ttagtcaaag accccttaac ccgcaaaatt gatcctgact atatccgcgc
     7261 cacacgcaag tttagagacc acttctttgt cctcaccaac ggtgaacatg aaggcagaag
     7321 gggagtaaat cgcatcgttg aacgggcatt tcgcaatgtt gaagccaaag aggaaacaag
     7381 ctatttacct ggtttagcag cagggggtgt gcaatggcag acagataatg gccaaatttc
     7441 ccatcccggt gttagccaag cagaactcga tttccttgcc acagtgccag atttaattgg
     7501 tcaaagttta ggacaatttt ttactaaata tgttgatatt tttcccgctg agcttcaacc
     7561 tgagctgatc catgcttctg tcatgcgccc tatagtgagt cgtattacaa ttcactggcc
     7621 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     7681 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     7741 caacagttgc gcagcctgaa tggcgaatgg acgcgccctg tagcggcgca ttaa
//
